Transcription factor coactivator protein, p/CIP

ABSTRACT

The present invention provides a substantially purified nucleic acid molecule encoding p/CIP, which regulates the activity of CBP/p300 dependent transcription factors. The invention also provides substantially purified p/CIP and peptide portions of p/CIP. The invention also provides methods of selectively inhibiting signal transduction pathways using a peptide portion of p/CIP or a nucleic acid molecule encoding such a peptide portion.

This application claims the benefit of priority of U.S. provisionalapplication 60/049,452, filed Jun. 12, 1997, the entire contents ofwhich are herein incorporated by reference.

ACKNOWLEGMENT

This invention was made with government support under grant numberDK39949-14A1 awarded by the National Institutes of Health and grantnumber CA52599-07 awarded by the National Cancer Institute. Thegovernment has certain rights in the invention.

SEQUENCE LISTING

A sequence listing appendix is submitted under 1.821(c) and isincorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to molecular biology andbiochemistry and more specifically to a coactivator protein, p/CIP,which is involved in regulating gene expression by CBP/p300-dependenttranscription factors, and to methods of using the coactivator proteinto selectively regulate gene expression.

2. Background Information

Regulation of gene expression is mediated by sequence-specifictranscription factors that bind to target genes and activate or represstranscription. Many of these factors are controlled by extracellularsignals that switch the factors between inactive and active states. Suchsignals can result in post-translational modification as observed, forexample, with the members of the STAT family of transcription factors,or can result in ligand-induced conformational changes as observed, forexample, with members of the nuclear receptor family of transcriptionfactors.

Coactivator proteins have been identified that are recruited to theactive forms of such transcription factors and are required for theirtranscriptional effects. The coactivators, CBP and p300, for example,serve essential roles in transcriptional activation by several classesof regulated transcription factors, including nuclear receptors, STATfactors, AP-1 proteins, NF-KB and CREB. In addition, a more recentlydiscovered family of proteins, termed nuclear receptor coactivator(NCoA) proteins, can interact with various nuclear receptors in aligand-dependent manner and also can interact with CBP and p300.

Two members of the NCoA family of proteins, NCoA-1 and NCoA-2, appear tohave relatively selective roles in mediating the transcriptional effectsof nuclear receptors. Evidence indicates, however, that additionalfactors are required for the transcriptional activities of manyCBP-dependent transcription factors, including STAT 1, AP-1 and CREB,and that complexes containing such coactivators, for example, CBP/p300and NCoA, are involved in transmitting an activation signal to thepromoter. Since CBP and p300-containing complexes appear to be limitingin cells, antagonistic interactions between signaling pathways can bedue, at least in part, to competition for these complexes. Thus, a needexists to identify different classes of transcription factors that areregulated by a CBP-containing complex and to identify the coactivatorproteins involved in such complexes. The present invention satisfiesthis need and provides related advantages as well.

SUMMARY OF THE INVENTION

The present invention provides a substantially purified nucleic acidmolecule having a nucleotide sequence encoding a transcriptionalcoactivator protein, designated p/CIP, which binds to CBP/p300-dependenttranscription factors and regulates their activity. For example, theinvention provides a substantially purified nucleic acid molecule havingthe nucleotide sequence shown in FIG. 1, which encodes murine p/CIP, anda nucleotide sequence complementary to that shown in FIG. 1.

The invention also provides a substantially purified nucleic acidmolecule encoding an active fragment of a p/CIP polypeptide, which has anucleotide sequence encoding substantially the same amino acid sequenceas a portion of a p/CIP polypeptide. Such a nucleic acid molecule canencode, for example, an active fragment including a CBP interactiondomain, such as a fragment having about amino acids 758 to 1115 of p/CIPshown in FIG. 1, or a nuclear receptor interaction domain, such as afragment having about amino acids 591 to 803 or about amino acids 680 to740 of p/CIP shown in FIG. 1.

Further provided herein is a substantially purified nucleic acidmolecule having a nucleotide sequence encoding a full length mouseNCoA-2 protein, which is related to p/CIP. The invention also provides asubstantially purified NCoA-2 active fragment, having a nucleotidesequence encoding substantially the same amino acid sequence as aportion of a NCoA-2 polypeptide. Such a NCoA-2 active fragment caninclude, for example, a nuclear receptor interaction domain.

The invention also provides vectors comprising a nucleic acid moleculeof the invention and host cells containing such vectors. In addition,the invention provides a substantially purified p/CIP nucleotidesequence having at least about 14 consecutive nucleotides of thenucleotide sequence shown in FIG. 1, or a nucleotide sequencecomplementary thereto.

The present invention also provides a substantially purified p/CIPpolypeptide, which can bind to a CBP/p300-dependent transcription factorand regulate its activity. For example, the invention provides asubstantially purified p/CIP polypeptide having substantially the sameamino acid sequence as p/CIP shown FIG. 1. The invention additionallyprovides a substantially purified p/CIP active fragment havingsubstantially the same amino acid sequence as a portion of a p/CIPpolypeptide. A particularly useful p/CIP active fragment can include,for example, a CBP interaction domain or a nuclear receptor interactiondomain, or can be an portion of a p/CIP polypeptide useful for elicitingproduction of an antibody that specifically binds to p/CIP.

The invention further provides a substantially purified NCoA-2polypeptide having substantially the same amino acid sequence as aminoacid sequence shown in FIG. 2a. Active fragments of a NCoA-2 polypeptideof the invention also are provided herein.

The invention also provides anti-p/CIP antibodies that specifically bindto p/CIP, as well as p/CIP-binding fragments of such antibodies. Theinvention further provides anti-NCoA-2 antibodies and antigen bindingfragments thereof. In addition, the invention provides cell linesproducing anti-p/CIP antibodies or anti-NCoA-2 antibodies.

The present invention further provides methods of identifying aneffective agent that alters the association of p/CIP or NCoA-2polypeptide with a second protein, such as a nuclear receptor or a CBP,which associates with the p/CIP or NCoA-2 polypeptide in vitro or invivo. The method includes the steps of contacting a p/CIP or NCoA-2polypeptide with an agent under conditions that allow the p/CIP orNCoA-2 polypeptide to associate with the second protein, and detectingan altered association of the p/CIP or NCoA-2 polypeptide with thesecond protein. An agent that alters the association of p/CIP, forexample, with a second protein can be a peptide, a polypeptide, apeptidomimetic or an organic molecule, such an effective agent beinguseful, for example, for modulating the level of transcription in acell. For example, a peptide portion of p/CIP comprising a helicalleucine-rich, charged domain (LCD), can inhibit the transcriptionalactivity of one type of nuclear receptor, such as the retinoic acidreceptor, but not of a second, related nuclear receptor such as theestrogen receptor, whereas a second LCD of p/CIP can inhibit signaltransduction induced by interferon γ, but not signal transductioninduced by retinoic acid. Thus, selected peptide portions of p/CIP or ofNCoA-2 can be valuable for regulating gene expression in a cell, andthese and other effective agents can have therapeutic efficacy.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the nucleotide sequence (SEQ ID 1) and deduced amino acidsequence (SEQ ID 2) of p/CIP.

FIGS. 2a to 2 d provide a characterization of p/CIP and a related memberof the NCoA family, NCoA-2. (SEQ ID 3).

FIGS. 2a to 2 d provide a characterization of p/CIP (SEQ ID 2) and arelated member of the NCoA family, NCoA-2 (SEQ ID 3). The conservedbHLH, PAS “A” domain, the nuclear receptor interaction domains and theminimal nuclear receptor and CBP interaction domains are boxed, andrepeat motifs involved in critical interactions are bracketed.

FIGS. 2b and 2 c provide western blot analyses of total cell extractsfor p/CIP, NCoA-1 and NCoA-2 in various tissues and cell lines, showingwidespread expression of all three proteins, although relative levelsdiffer.

FIG. 2d provides schematic diagrams showing regions of homology of p/CIPwith NCoA-1 and NCoA-2. The asterisks refer to the repeated peptidemotifs that appear to be of functional importance (see FIGS. 6 and 7).

FIG. 3 shows the results of biochemical analysis of p/CIP and NCoAFactors.

FIG. 3a demonstrates interactions between recombinant GST proteins andNCoA proteins from HeLa whole cell extracts detected using an anti-p/CIPantibody (left) or an anti-NCoA-1 antibody (right).

FIG. 3b (left) shows co-immunoprecipitation of CBP/p300 and p/CIP.Anti-p/CIP, anti-NCoA-1 or anti-NCoA-2 IgG was incubated with HeLa wholecell extracts and immunocomplexes were separated by SDS-PAGE and probedusing anti-CBP/p300 IgG. FIG. 3b (right) shows the detection of CBP/p300in supernatant following immunodepletion of whole cell extracts withspecific anti-NCoA antibodies.

FIG. 3c shows the results of yeast two-hybrid assays mapping regions ofinteraction between p/CIP and the CBP C-terminus (aa 2058-2170) orliganded estrogen receptor (LBD).

FIG. 3d demonstrates that a common nuclear receptor interaction domainis found in p/CIP, NCoA-1 and NCoA-2 by yeast two-hybrid assay. Ligands(+) were estradiol (10⁻⁶ M), Triac (10⁻⁶ M) or retinoic acid (10⁻⁶ M)

FIG. 3e shows p/CIP, NCoA-1 or NCoA-2 interactions with nuclearreceptors in vitro. Recombinant GST-nuclear receptor proteins wereincubated with whole cell extract in the presence(+) or absence (−) ofligand, then western blot analysis was performed using p/CIP-, NCoA-1-or NCoA-2-specific IgG.

FIG. 3f shows the results of transcription activation studies, in whichreporter genes containing the minimal prolactin promoter (P-36luciferase), alone, or two copies of the indicated response elements,and plasmids expressing p/CIP, NCoA-1 or NCoA-2 were transfected intoHeLa cells in the presence of the corresponding ligand. The effect ofvarying amounts of plasmid expressing GAL4 (1-147), GAL4-NCoA-1 orGAL4-p/CIP fusion proteins on a minimal (UAS)₆-dependent reporter areshown in the right panel.

FIG. 4 demonstrates a role of P/CIP in the function of CBP-dependenttranscription factors.

FIG. 4a shows the effect of microinjection of affinity-purifiedanti-p/CIP IgG on ligand-dependent gene activation by RAR in Rat-1cells.

FIG. 4b shows experiments as in FIG. 4a, but performed using minimalpromoters with four copies of the estrogen (ERE), thyroid hormone (TRE)or progesterone (PRE) receptor response elements.

FIG. 4c demonstrates that both CBP and p/CIP expression vectors arerequired to rescue anti-p/CIP IgG inhibition of RAR-dependent geneactivation.

FIG. 4d shows the effect of expression of the p/CIP core CBP interactiondomain (947-1084) on RAR dependent transcription (left) or on SP-1 orCMV dependent transcription (right).

FIG. 4e shows the effect of anti-p/CIP IgG (α p/CIP) on an interferon γdependent promoter (GAS/LacZ)¹² (left) and the effect of p/CIP (aa947-1084) on interferon γ stimulated transcriptions and failure of CBPexpression vector to rescue this inhibition (right).

FIG. 4f shows the effect of anti-NCoA-1 IgG (α 1) on GAS andcAMP-dependent (2×CRE) promoters. All were performed at least threeseparate times, with >200 cells injected; error bars are ±2×SEM.

FIG. 5 demonstrates a role for NCoA-1 and NCoA-2 in nuclear receptorfunction.

FIG. 5a demonstrates that microinjection of affinity-purifiedanti-NCoA-1, but not of anti-NCoA-2, IgG blocked ligand-dependent geneactivation by RAR (left), but did not inhibit expression of either the6×SP-1 or CMV-driven promoters (right).

FIG. 5b shows experiments as in FIG. 5a, except using minimal promoterswith two copies of the estrogen (ERE) or T3R (TRE) response elementswith less profound effects upon progesterone (PRE) mediatedtranscription.

FIG. 5c demonstrates that anti-NCoA-1 IgG blocked retinoicacid-dependent activation of the RARE/LacZ reporter was not rescued byCMV expression vectors expressing p/CIP or CBP; however, expression wasfully rescued by CMV-NCoA-1 and b CMV-NCoA-2.

FIG. 5d shows photomicrographs of rhodamine-stained injected cells andthe corresponding protein of XGal staining

FIG. 6 shows the leucine-rich charged domains (LCD's) in p/CIP/NCoA/CBP.

FIG. 6a shows that a repeated leucine-rich domain is required forprotein-protein interactions between p/CIP, CBP, NCoA proteins andnuclear receptors. The sequence of some of these domains are noted (SEQID 2, aa 609-621, 670-684, 723-738, 1037-1049, 1058-1068; SEQ ID 3, aa636-648, 682-696, 739-753, 1057-1069, 1078-1088; SEQ ID 4-SEQ ID 9) withthe core hexapeptide motifs indicated by brackets. Helical wheels ofNCoA-1 LCD2 (SEQ ID 10) and CBP LCD6 (SEQ ID 11) are shown.

FIG. 6b shows that mutation of amino acids 70-73 in CBP (QLSELL (SEQ ID5, aa 8-13)→QLAAAA (SEQ ID 12)) resulted in a complete loss ofligand-dependent interaction with T3R.

FIG. 6c shows results of the yeast two-hybrid assay of interactionsbetween the NCoA-1 nuclear receptor interaction domains (aa 635-760)with nuclear receptors (left). Mutations of the LCD2 motif (RLHRLL (SEQID 5, aa 8-13)→RLAAAA (SEQ ID 12)) abolished ligand-dependentinteraction, while peptides encompassing LCD2 (37 amino acids “aa”)alone or LCD6 (59 amino acids) were sufficient for ligand-dependentinteraction (center). 24-mer peptides encompassing LCD1, LCD2 or acontrol peptide were tested for ability to inhibit binding of³⁵S-labeled NCoA interaction domain fragment (aa 635-760) to ligandedRAR with TTNPB (1 μM) (right)

FIGS. 6d and 6 e demonstrate the functional effect of plasmidsexpressing mutations in LCD2 (HRLL (SEQ ID 5, aa 10-13)→AAAA (SEQ ID 12,aa 3-6)) and LCD 3 (RYLL (SEQ ID 6, aa 8-11)→AAAA (SEQ ID 13)) of NCoA-1on resuce of inhibition by microinjected anti-NCoA-1 IgG (α-1) onretinoic dependent transcription (FIG. 6d) and on estrogen dependenttranscription (FIG. 6e).

FIG. 7 demonstrates that distinct helical motifs block transcriptionaleffects of specific signal transduction pathways.

FIG. 7a shows that a 19-mer peptide, corresponding to NCoA-1 LCD4, butnot a control peptide (CBP-622, control P), inhibits retinoic acidinduced, but not interferon γ induced gene expression.

FIG. 7b shows the effect of microinjection of the N-terminal 22 aminoacids of CBP (CBP N′-P1), a synthetic N-terminal CBP peptide, onretinoic acid and interferon gene activation events. A synthetic peptidecorresponding to the identical peptide lacking the eight amino terminalamino acids (CBP N′-P2) failed to inhibit interferon-dependent geneactivation events.

FIG. 7c shows, similar to FIG. 7b, that interferon γ inhibition ofretinoic acid-dependent activation of the RARE/LacZ reporter (rightpanel) was fully abolished by co-injection of the CBP N′-P1 peptide,which had no effect on retinoic acid dependent inhibition of theGAS/LacZ reporter by activated retinoic acid reporter.

FIG. 7d provides a model of p/CIP/CBP (p300) function, indicating thatseveral signal transduction pathways mediated by specific transcriptionfactors require a functional p/CIP, CBP/p300 complex, and potentiallyp/CAF, with each partner required, but not sufficient, to mediatetranscriptional effects. Nuclear receptor-specific requirements fordistinct protein-protein associations via specific LCD's is suggested.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a substantially purified nucleic acidmolecule encoding a transcriptional coactivator protein, designatedp/CIP (p300/CBP/co-integrator-associated Protein), which binds toCBP/p300-dependent transcription factors and regulates their activity.For example, the invention provides a substantially purified nucleicacid molecule having the nucleotide sequence shown in FIG. 1, whichencodes p/CIP, and a nucleotide sequence complementary to that shown inFIG. 1. As disclosed herein, p/CIP is a member of the NCoA (Nuclearreceptor Co-Activator) gene family and is involved in regulating thetranscriptional activities of various CBP-dependent transcriptionfactors, including STAT 1, AP-1 and CREB. In addition, the inventionprovides a substantially purified nucleic acid molecule encoding a fulllength murine NCoA-2 polypeptide having the amino acid sequence shown inFIG. 2a.

As used herein, the term “substantially purified,” when used inreference to a nucleic acid molecule of the invention, means that thenucleic acid molecule is relatively free from contaminating lipids,proteins, nucleic acids or other cellular material normally associatedwith a nucleic acid molecule in a cell. A substantially purified nucleicacid molecule of the invention can be obtained, for example, by chemicalsynthesis of the nucleotide sequence shown in FIG. 1 or by cloning themolecule using methods such as those disclosed in Example I.

As disclosed herein, CBP is present in a complex with p/CIP, which isrequired for transcriptional activity of nuclear receptors and otherCBP/p300-dependent transcription factors, including STAT and AP-1. Therelated nuclear receptor coactivator protein, NCoA-1, also isspecifically required for ligand-dependent gene activation by nuclearreceptors. p/CIP, NCoA-1, and CBP contain related leucine-rich chargedhelical interaction motifs that are required for receptor-specificmechanisms of gene activation. The disclosure of these leucine-richmotifs permits selective inhibition of distinct signal transductionpathways.

CBP and p300 are functionally conserved proteins that have intrinsicacetylase activity and serve essential roles in activation by a largenumber of regulated transcription factors, including nuclear receptors,CREB, AP-1, bHLH and STAT proteins (see, for example, Chakravarti etal., Nature 383:99-103 (1996); Kwok et al., Nature 370:223-226 (1994);Arias et al., Nature 370: 226-229 (1994); Eckner et al., Genes andDevel. 10(19): 2478-2490 (1996), each of which is incorporated herein byreference).

In addition to CBP and p300, a series of factors that exhibitligand-dependent and AF2-dependent binding to nuclear receptor C-terminihave been identified biochemically (see Halachmi et al., Science264:1455-1458 (1994); Cavailles et al., EMBO J. 14:3741-3751 (1995);Kurokawa et al., Nature 377:451-454 (1995)) and by expression cloning(see Lee et al., Nature 374:91-94 (1995); Le Douarin et al., EMBO J.14:2020-2033 (1995); Voegel et al., EMBO J. 15(14):3667-3675 (1996);Hong et al., Proc. Natl. Acad. Sci., USA 93:4948-4952 (1996)). Twohomologous factors, termed SRC-1/NCoA-1 and TIF-2/GRIP-1, increaseligand-dependent transcription by several nuclear receptors incotransfection assays and constitute a nuclear receptor coactivator(NCoA) gene family.

p/CIP is a NCoA/SRC family member that forms a complex with CBP in acell. Surprisingly, both p/CIP and NCoA-1 are required for the functionof nuclear receptors, while p/CIP, but not NCoA-1, is required forfunction of other CBP-dependent transcription factors. A series ofhelical leucine-rich, charged residue-rich domains (LCD's) within thesefactors serve as interaction motifs that are involved in assembly of acoactivator complex and that contribute to the specificity of nuclearreceptor activation events.

Studies of CBP, NCoA-1 and p/CIP have led to the identification of aseries of helical motifs that are required for NCoA/nuclear receptorinteraction, NCoA/CBP interaction and CBP/STAT interaction. Based on theidentification of these helical motifs, corresponding peptides have beendeveloped that, when injected into cells, selectively block signaling byretinoic acid, estrogen or interferon γ. These results demonstrate thatthe targeting of specific interaction motifs present in coactivatorcomplexes can result in highly selective effects on patterns of geneexpression. These observations further indicate that the utilization ofspecific interaction motifs by nuclear receptors can be altered bydifferent classes of ligands, resulting in selective activities that canbe of therapeutic benefit. Thus, the invention provides methods ofidentifying agents that modulate the activity of specific classes oftranscription factors.

As disclosed herein, the cloned p/CIP cDNA is a novel member of the NCoAgene family. Like NCoA-1 (SRC-1) and NCoA-2 (TIF2, GRIP2), p/CIPinteracts with several nuclear receptors in a ligand-dependent manner.Analysis of p/CIP, NCoA-1 and NCoA-2 has led to identification of aseries of helical interaction motifs that mediate interactions betweenNCoA proteins and nuclear receptors, and a separate series of helicalmotifs that mediate interactions between NCoA proteins and CBP (see, forexample, FIG. 2a). Mutations within these motifs reduce the ability ofthe coactivator to mediate transcriptional activation. Remarkably,microinjection of peptides cornerbanding to specific helical motifsexert selective inhibitory effects on transcription by different classesof transcription factors. Using this strategy, effective agents such aspeptides have been identified that selectively block STAT 1 activity butnot nuclear receptor-dependent transcription. In addition, effectiveagents have been identified that selectively block retinoic acidreceptor activity but not STAT 1 activity. These agents have been usedto demonstrate that inhibitory effects of interferon γ on retinoic acidtranscription involve the CBP/p/CIP coactivator complex.

The invention also provides vectors comprising a nucleic acid moleculeof the invention and host cells containing such vectors. In addition,the invention provides nucleotide sequences that bind to a nucleic acidmolecule of the invention, such nucleotide sequences being useful, forexample, as probes, which can identify the presence of a nucleic acidmolecule encoding p/CIP in a sample or as antisense molecules, which caninhibit the expression of a nucleic acid molecule encoding a p/CIP.

A substantially purified nucleic acid molecule of the invention isexemplified by the nucleotide sequence shown in FIG. 1, which encodesp/CIP protein, also shown in FIG. 1. Due to the degeneracy of thegenetic code and in view of the disclosed amino acid sequence of a p/CIPprotein, additional nucleic acid molecules of the invention would bewell known to those skilled in the art. Such nucleic acid molecules havea nucleotide sequence that is different from that shown in FIG. 1 but,nevertheless, encode the amino acid sequence shown in FIG. 1. Thus, theinvention provides a substantially purified nucleic acid moleculecomprising a nucleotide sequence encoding the amino acid sequence ofmurine p/CIP as shown in FIG. 1. Similarly, the invention provides asubstantially purified nucleic acid molecule encoding a full lengthNCoA-2 polypeptide having the amino acid sequence shown in FIG. 2a.

As used herein, reference to “a nucleic acid molecule encoding p/CIP”indicates 1) the polynucleotide sequence of one strand of a doublestranded DNA molecule comprising the nucleotide sequence that codes, forexample, for p/CIP and can be transcribed into an RNA that encodes thecoactivator, or 2) an RNA molecule, which can be translated, forexample, into p/CIP. It is recognized that a double stranded DNAmolecule also comprises a second polynucleotide strand that iscomplementary to the coding strand and that the disclosure of apolynucleotide sequence comprising a coding sequence necessarilydiscloses the complementary polynucleotide sequence. Accordingly, theinvention provides polynucleotide sequences, including, for example,polydeoxyribonucleotide or polyribonucleotide sequences that arecomplementary to the nucleotide sequence shown in FIG. 1 or to a nucleicacid molecule encoding p/CIP having the amino acid sequence shown inFIG. 1.

As used herein, the term “polynucleotide” is used in its broadest senseto mean two or more nucleotides or nucleotide analogs linked by acovalent bond. The term “oligonucleotide” also is used herein to meantwo or more nucleotides or nucleotide analogs linked by a covalent bond,although those in the art will recognize that oligonucleotides generallyare less than about fifty nucleotides in length and, therefore, are asubset within the broader meaning of the term “polynucleotide.”

In general, the nucleotides comprising a polynucleotide are naturallyoccurring deoxyribonucleotides, such as adenine, cytosine, guanine orthymine linked to 2′-deoxyribose, or ribonucleotides such as adenine,cytosine, guanine or uracil linked to ribose. However, a polynucleotidealso can comprise nucleotide analogs, including non-naturally occurringsynthetic nucleotides or modified naturally occurring nucleotides. Suchnucleotide analogs are well known in the art and commercially available,as are polynucleotides containing such nucleotide analogs (Lin et al.,Nucl. Acids Res. 22:5220-5234 (1994); Jellinek et al., Biochemistry34:11363-11372 (1995); Pagratis et al., Nature Biotechnol. 15:68-73(1997)). The covalent bond linking the nucleotides of a polynucleotidegenerally is a phosphodiester bond. However, the covalent bond also canbe any of numerous other bonds, including a thiodiester bond, aphosphorothioate bond, a peptide-like bond or any other bond known tothose in the art as useful for linking nucleotides to produce syntheticpolynucleotides (see, for example, Tam et al., Nucl. Acids Res.22:977-986 (1994); Ecker and Crooke, BioTechnology 13:351360 (1995)).

Where it is desired to synthesize a polynucleotide of the invention, theartisan will know that the selection of particular nucleotides ornucleotide analogs and the covalent bond used to link the nucleotideswill depend, in part, on the purpose for which the polynucleotide isprepared. For example, where a polynucleotide will be exposed to anenvironment containing substantial nuclease activity, the artisan willselect nucleotide analogs or covalent bonds that are relativelyresistant to the nucleases. A polynucleotide comprising naturallyoccurring nucleotides and phosphodiester bonds can be chemicallysynthesized or can be produced with recombinant DNA methods using anappropriate polynucleotide as a template. In comparison, apolynucleotide comprising nucleotide analogs or covalent bonds otherthan phosphodiester bonds generally will be chemically synthesized,although an enzyme such as T7 polymerase can incorporate certain typesof nucleotide analogs and, therefore, can be used to produce such apolynucleotide recombinantly from an appropriate template (Jellinek etal., supra, 1995).

The invention also provides nucleotide sequences that can bind to anucleic acid molecule encoding p/CIP. Such nucleotide sequences areuseful, for example, as probes, which can hybridize to a nucleic acidmolecule encoding a p/CIP and allow the identification of the nucleicacid molecule in a sample. A nucleotide sequence of the invention ischaracterized, in part, in that it is at least nine nucleotides inlength, such sequences being particularly useful as primers for thepolymerase chain reaction (PCR), and can be, for example, at leastfourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty ortwenty-one nucleotides in length. If desired, a nucleotide sequence ofthe invention can have at least twenty-five, thirty, thirty-five, fortyor fifty nucleotides of the nucleotide sequence shown in FIG. 1. Such anucleotide sequence of the invention is useful as a hybridization probeor as a primer for PCR and can be used, for example, to identifyhomologous nucleic acid molecules encoding p/CIP proteins in othereukaryotes, particularly other mammals, including humans.

As disclosed herein, p/CIP is a member of the NCoA protein family and,therefore, shares conserved structural regions with other members ofthis family. Thus, a nucleic acid molecule encoding p/CIP shares regionsof substantial homology with a nucleic acid molecule encoding an NCoAprotein such as NCoA-2. However, a comparison of the nucleic acidmolecules encoding p/CIP and NCoA-2, for example, also will revealnucleotide sequences that are unique to p/CIP, such nucleotide sequencesbeing encompassed within the invention.

A substantially purified nucleotide sequence of the invention cancomprise a portion of a coding sequence of a nucleic acid moleculeencoding p/CIP or of a sequence complementary thereto, depending on thepurpose for which the nucleotide sequence is to be used. In addition, amixture of a coding sequence and its complementary sequence can beprepared and, if desired, can be allowed to anneal to produce doublestranded oligonucleotides. The invention also provides antisense nucleicacid molecules, which are complementary to a nucleic acid moleculeencoding p/CIP and can bind to and inhibit the expression of the nucleicacid molecule.

A nucleic acid molecule of the invention, including an antisensemolecule, can be introduced into a cell by methods of transfection, orcan be contained in a plasmid or viral vector, which can be introducedinto the cell, such that the nucleic acid molecule is stably ortransiently expressed (see, for example, Sambrook et al., MolecularCloning: A laboratory manual (Cold Spring Harbor Laboratory Press 1989);Ausubel et al., Current Protocols in Molecular Biology (Green Publ., NY1994), each of which is incorporated herein by reference). Accordingly,the invention provides vectors comprising a nucleic acid molecule of theinvention and host cells, which are appropriate for maintaining suchvectors. Vectors, which can be cloning vectors or expression vectors,are well known in the art and commercially available. An expressionvector comprising a nucleic acid molecule of the invention, which canencode a p/CIP or can be an antisense molecule, can be used to expressthe nucleic acid molecule in a cell.

In general, an expression vector contains the expression elementsnecessary to achieve, for example, transcription of the nucleic acidmolecule, although such elements also can be inherent to the nucleicacid molecule cloned into the vector. In particular, an expressionvector contains or encodes a promoter sequence, which can provideconstitutive or, if desired, inducible expression of a cloned nucleicacid sequence, a poly-A recognition sequence, and a ribosome recognitionsite, and can contain other regulatory elements such as an enhancer,which can be tissue specific. The vector also contains elements requiredfor replication in a procaryotic or eukaryotic host system or both, asdesired. Such vectors, which include plasmid vectors and viral vectorssuch as bacteriophage, baculovirus, retrovirus, lentivirus, adenovirus,vaccinia virus, semliki forest virus and adeno-associated virus vectors,are well known and can be purchased from a commercial source (Promega,Madison Wis.; Stratagene, La Jolla Calif.; GIBCO/BRL, Gaithersburg Md.)or can be constructed by one skilled in the art (see, for example, Meth.Enzymol., Vol. 185, D. V. Goeddel, ed. (Academic Press, Inc., 1990);Jolly, Canc. Gene Ther. 1:51-64 (1994); Flotte, J. Bioenerg. Biomemb.25:37-42 (1993); Kirshenbaum et al., J. Clin, Invest 92:381-387 (1993),which is incorporated herein by reference).

A nucleic acid molecule, including a vector, can be introduced into acell by any of a variety of methods known in the art (Sambrook et al.,supra, 1989, and in Ausubel et al., Current Protocols in MolecularBiology, John Wiley and Sons, Baltimore, Md. (1994), which isincorporated herein by reference). Such methods include, for example,transfection, lipofection, microinjection, electroporation and infectionwith recombinant vectors or the use of liposomes.

Introduction of a nucleic acid molecule by infection with a viral vectoris particularly advantageous in that it can efficiently introduce thenucleic acid molecule into a cell ex vivo or in vivo. Moreover, virusesare very specialized and typically infect and propagate in specific celltypes. Thus, their natural specificity can be used to target the nucleicacid molecule contained in the vector to specific cell types. Viral ornon-viral vectors also can be modified with specific receptors orligands to alter target specificity through receptor mediated events.

A nucleic acid molecule also can be introduced into a cell using methodsthat do not require the initial introduction of the nucleic acidmolecule into a vector. For example, a nucleic acid molecule encoding ap/CIP can be introduced into a cell using a cationic liposome, whichalso can be modified with specific receptors or ligands as describedabove (Morishita et al., J. Clin Invest., 91:2580-2585 (1993), which isincorporated herein by reference; see, also, Nabel et al., supra,1993)). In addition, a nucleic acid molecule can be introduced into acell using, for example, adenovirus-polylysine DNA complexes (see, forexample, Michael et al., J. Biol. Chem., 268:6866-6869 (1993), which isincorporated herein by reference). Other methods of introducing anucleic acid molecule into a cell such that the encoded p/CIP orantisense nucleic acid molecule can be expressed are well known (see,for example, Goeddel, supra, 1990).

Selectable marker genes encoding, for example, a polypeptide conferringneomycin resistance (Neo^(R)) also are readily available and, whenlinked to a nucleic acid molecule of the invention or incorporated intoa vector containing the nucleic acid molecule, allow for the selectionof cells that have incorporated the nucleic acid molecule. Otherselectable markers such as that conferring hygromycin, puromycin orZEOCIN (Invitrogen, Carlsbad Calif.) resistance are known to those inthe art of gene transfer as markers useful for identifying cellscontaining the nucleic acid molecule, including the selectable markergene.

A “suicide” gene also can be incorporated into a vector so as to allowfor selective inducible killing of a cell containing the gene. A genesuch as the herpes simplex virus thymidine kinase gene (TK) can be usedas a suicide gene to provide for inducible destruction of such cells.For example, where it is desired to terminate the expression of anintroduced nucleic acid molecule encoding p/CIP or an antisense p/CIPnucleic acid molecule in cells containing the nucleic acid molecule, thecells can be exposed to a drug such as acyclovir or gancyclovir, whichcan be administered to an individual.

Numerous methods are available for transferring nucleic acid moleculesinto cultured cells, including the methods described above. In addition,a useful method can be similar to that employed in previous human genetransfer studies, where tumor infiltrating lymphocytes (TILs) weremodified by retroviral gene transduction and administered to cancerpatients (Rosenberg et al., New Engl. J. Med. 323:570-578 (1990); see,also, Anderson et al., U.S. Pat. No. 5,399,346, issued Mar. 21, 1995,each of which is incorporated herein by reference).

The present invention also provides a substantially purified p/CIPpolypeptide, which forms a complex with CBP/p300 in a cell and regulatesCBP/p300-dependent transcriptional activity. A p/CIP polypeptide of theinvention is exemplified herein by murine p/CIP, which is a protein ofabout 152 kDa that has a conserved amino-terminal basic helix-loop-helixdomain, PAS “A” domain, a serine/threonine-rich region and acarboxy-terminal glutamine-rich region. Murine p/CIP is related toSRC-1/NCoA-1 and NCoA-2/TIF-2, showing overall amino acid identity of31% and 36%, respectively. As disclosed herein, p/CIP is required fortranscriptional activation by CBP-dependent transcription factors,including nuclear receptors such as the retinoic acid receptor, estrogenreceptor, thyroid receptor and progesterone receptor, and otherCBP-dependent transcription factors such as STAT 1 (see Example I).

Thus, the present invention provides a substantially purified p/CIPpolypeptide. Such a polypeptide can have, for example, substantially thesame amino acid sequence as murine p/CIP shown in FIG. 1 (see, also,FIG. 2a). Further provided herein is a substantially purified p/CIPactive fragment having substantially the same amino acid sequence as aportion of a p/CIP polypeptide. Such an active fragment can include, forexample, a CBP interaction domain or a nuclear receptor interactiondomain. LCD peptide portions of p/CIP also are provided (see Example I).

The term “substantially purified,” as used herein in reference to apolypeptide or fragment thereof, means that the polypeptide orpolypeptide fragment is relatively free from contaminating lipids,proteins, nucleic acids or other cellular material normally associatedwith a polypeptide in a cell.

As used herein, the term “p/CIP” or “p/CIP polypeptide” means thepolypeptide referred to herein as “p300/CBP/CoIntegrator-associatedProtein” and includes the murine p/CIP polypeptide shown in FIG. 1. Asdescribed above, murine p/CIP displays homology to SRC-1/NCoA-1 andNCoA-2/TIF-2, sharing basic helix-loop-helix domains, a PAS “A” domain,serine/threonine-rich region and glutamine-rich region with NCoA-1 andNCoA-2.

The term p/CIP encompasses murine p/CIP and is intended to includerelated polypeptides having substantial amino acid sequence similarityto this polypeptide. Such related polypeptides will exhibit greatersequence similarity to p/CIP than to SRC-1/NCoA-1 or to NCoA-2/TIF-2 andinclude alternatively spliced forms of p/CIP and isotype variants of theamino acid sequence shown in FIG. 1. The term p/CIP also encompasseshomologous polypeptides obtained from different mammalian species, suchas a human homolog of the murine p/CIP polypeptide disclosed in FIG. 1.A p/CIP polypeptide generally has an amino acid identity of greater thanabout 40%, preferably greater than about 50%, more preferably greaterthan about 60%, and can have an amino acid identity of greater thanabout 70%, 75%, 80%, 85%, 90% or 95% with the murine p/CIP amino acidsequence disclosed in FIG. 1.

As used herein, the term “substantially the same amino acid sequence,”when used in reference to a p/CIP amino acid sequence, is intended tomean the amino acid sequence shown in FIG. 1, or a similar,non-identical sequence that is considered by those skilled in the art tobe a functionally equivalent nucleotide or amino acid sequence. Thus, apolypeptide that has substantially the same amino acid sequence as ap/CIP polypeptide can have one or more modifications such as amino acidadditions, deletions or substitutions relative to the amino acidsequence shown in FIG. 1, provided that the p/CIP polypeptide retains atleast one biological activity of a native p/CIP polypeptide.

Therefore, it is understood that modifications can be made withoutdestroying the biological function of a p/CIP polypeptide. Also, forexample, genetically engineered variants of p/CIP either alone or fusedto heterologous proteins that retain at least one measurable activity inbinding to a CBP protein, binding to a nuclear receptor, activity inretinoic acid, estrogen, thyroid or progesterone dependenttranscription, activity in other CBP-dependent transcription, or otherinherent biological activity fall within the definition of a p/CIPpolypeptide.

It is understood that modifications of primary amino acid sequence canresult in polypeptides which have substantially equivalent, enhanced orreduced function as compared to the murine p/CIP sequence set forth inFIG. 1. These modifications can be deliberate, as through site-directedmutagenesis, or can be accidental such as through mutation in hostsharboring a p/CIP encoding nucleic acid molecule. All such modifiedpolypeptides are included in the definition of a p/CIP polypeptide aslong as at least one biological function of a p/CIP polypeptide isretained. Further, various molecules can be attached to a p/CIPpolypeptide including, for example, other polypeptides, carbohydrates,lipids, or chemical moieties using methods well known in the art. Suchmodifications are included within the term “p/CIP polypeptide,” asdefined herein.

Further provided herein is an isolated active fragment of a p/CIPpolypeptide, which includes substantially the same amino acid sequenceas a portion of a p/CIP polypeptide. As used herein, the term “activep/CIP fragment” means a peptide or polypeptide which has substantiallythe same amino acid sequence as a portion of a p/CIP polypeptide,provided that the fragment retains at least one biological activity of ap/CIP polypeptide. As defined herein, an active fragment generally hasan amino acid sequence of about 15 to about 400 contiguous residues andcan have, for example, an amino acid sequence of at least about 18, 20,25, 30, 35, 40, 50, 100, 150, 200, 250, 300, 350 or 400 contiguousresidues. A particularly useful active fragment has from about 80 toabout 150 amino acids. A biological activity of a p/CIP polypeptide thatis retained by an active p/CIP fragment can be, for example, measurableactivity in binding to a CBP protein, binding to a nuclear receptor,activity in retinoic acid, estrogen, thyroid or progesterone dependenttranscription, activity in other CBP-dependent transcription, or otherinherent biological activity.

An isolated p/CIP active fragment of the invention can include, forexample, a CBP interaction domain. Such a CBP-binding active fragmentcan have, for example, an amino acid sequence that is identical orsubstantially the same as a portion of p/CIP shown in FIG. 1, forexample, substantially the same as about amino acids 758 to 1115 ofp/CIP, about amino acids 947 to 1084 of p/CIP, or about amino acids 163to 610 of p/CIP shown in FIG. 1. Additional p/CIP active fragmentshaving a CBP interaction domain can readily be identified, for example,using yeast two-hybrid assays or microinjection assays, as set forth inExample I. As disclosed herein, such an active fragment can blockCDP-dependent gene activation, for example, interferon-γ or TPAstimulated gene activation or retinoic acid dependent gene activation.

A substantially purified p/CIP active fragment of p/CIP can include anuclear receptor interaction domain. Such a nuclear receptor-bindingactive fragment of p/CIP can have, for example, an amino acid sequencethat is identical or substantially the same as a portion of p/CIP shownin FIG. 1, and can bind a nuclear receptor such as the estrogenreceptor, for example, in a ligand-dependent manner. An example of ap/CIP active fragment having a nuclear receptor interaction domain is afragment having substantially the same amino acid sequence as aboutamino acids 591 to 803 of p/CIP or about amino acids 680 to 740 of p/CIPshown in FIG. 1.

Also provided herein is a novel member of the nuclear receptorco-activator family designated NCoA-2. As disclosed herein, murineNCoA-2 is a polypeptide of about 160 kDa that interacts with a 100 aminoacid region in the carboxy termini of CBP (amino acids 2058-2170), aswell as with the liganded estrogen receptor. Thus, the inventionprovides a substantially purified NCoA-2 polypeptide havingsubstantially the same amino acid sequence as the amino acid sequenceshown in FIG. 2a. In addition, the invention provides a substantiallypurified NCoA-2 active fragment having substantially the same amino acidsequence as a portion of a NCoA-2 polypeptide. An active fragment of aNCoA-2 polypeptide can include, for example, a nuclear receptorinteraction domain.

As used herein, the term “NCoA-2” or “NCoA-2 polypeptide” is intended tomean a polypeptide having substantial similarity to the murine NCoA-2polypeptide shown in FIG. 2a. Like a p/CIP polypeptide, a NCoA-2polypeptide has a basic helix-loop-helix domain, a PAS “A” domain, aserine/threonine-rich region and a glutamine-rich region.

The term NCoA-2 encompasses murine NCoA-2 and is intended to includerelated polypeptides having substantial amino acid sequence similarityto this polypeptide. Such related polypeptides will exhibit greatersequence similarity to NCoA-2 than to SRC-1/NCoA-1 or to p/CIP andinclude alternatively spliced forms of NCoA-2 and isotype variants ofthe amino acid sequences shown in FIG. 2a. The term NCoA-2 alsoencompasses homologous polypeptides obtained from different mammalianspecies, although the human TIF-2 and GRIP-1 polypeptides described inVoegel et al., EMBO J. 15:3667-3675 (1996) and Hong et al., Proc. Natl.Acad. Sci. USA 93:4948-4952 (1996), each of which is incorporated byreference herein, are explicitly excluded from the term NCoA-2polypeptide as defined herein. A NCoA-2 polypeptide generally has anamino acid sequence having an amino acid identity of greater than about70%, preferably greater than about 75%, more preferably greater thanabout 80%, and can have an amino acid identity of greater than about85%, 90% or 95% with the murine NCoA-2 amino acid sequence disclosed inFIG. 2a.

An active fragment of a p/CIP or NCoA-2 polypeptide can be produced byany of several methods well known in the art. For example, an activefragment of the invention can be produced by enzymatic cleavage of ap/CIP polypeptide using a proteolytic enzyme such as trypsin,chymotrypsin or the like, or a combination of such enzymes. Theresulting enzymatic digestion subsequently can be purified using wellknown methods. An active fragment also can be produced using methods ofsolution or solid phase peptide synthesis or can be expressed from anucleic acid molecule such as a portion of the coding region of thenucleic acid sequence shown in FIG. 1, or can be purchased from acommercial source.

The invention also provides an LCD peptide portion of p/CIP, whichincludes a helical leucine-rich, charged domain (LCD) and which caninhibit the transcriptional activity of one type of nuclear receptor,such as the retinoic acid receptor, but not of a second, related nuclearreceptor such as the estrogen receptor. An LCD peptide portion of p/CIPalso can selectively inhibit signal transduction induced by interferon γwithout inhibiting signal transduction induced by retinoic acid. Thus,an LCD peptide portion of p/CIP or of another NCoA can be useful forregulating gene expression in a cell.

An LCD peptide portion of p/CIP or NCoA-2 is characterized, in part, ascontaining one or more copies of the consensus core sequence, LXXLL,where L is leucine and X is independently selected to be any amino acid.Preferably, an LCD peptide portion contains at least three copies of theconsensus core sequence LXXLL. An LCD peptide portion p/CIP can include,for example, one or more of the following amino acid sequences:KGHKKLLQLLTCS (SEQ ID 2, aa 609-621), LLQEKHRILHKLLQN (SEQ ID 2, aa670-684), KKENNALLRYLLDRDD (SEQ ID 2, aa 723-738), LRNSLDDLLGPPS (SEQ ID2, aa 1037-1049) or RALLDQLHTFL (SEQ ID 2, aa 1058-1068). An LCD peptideportion of NCoA-2 can include, for example, one or more of the followingamino acid sequences: KGQTKLLQLLTTK (SEQ ID 3, 636-648), SLKEKHKILHRLLQD(SEQ ID 3, 682-696), KKENALLRYLLDKDD (SEQ ID 3, 739-753), FGSSPDDLLCPHP(SEQ ID 3, aa 1057-1069) or GALLDQLYLAL (SEQ ID 3, 1078-1088). An LCDpeptide portion of p/CIP or of NCoA-2 can be a helical domain withamphipathic characteristics and can have a length of eight, nine, ten,twelve, fourteen, sixteen, twenty, forty, sixty, eighty or moreresidues.

As used herein, the term “amino acid” includes both amino acids andamino acid equivalents. An amino acid equivalent is a compound whichdeparts from the structure of a naturally occurring amino acid, butwhich has substantially the structure of an amino acid, such that it canbe substituted within a peptide or protein which retains its biologicalactivity. Thus, for example, amino acid equivalents can include aminoacids having side chain modifications or substitutions, and also caninclude related organic acids, amides or the like. Amino acidequivalents include amino acid mimetics, which are those structureswhich exhibit substantially the same spatial arrangement of functionalgroups as amino acids but do not necessarily have both the α-amino andα-carboxyl groups characteristic of amino acids.

The invention also provides anti-p/CIP antibodies and anti-murine NCoA-2antibodies, as well as antigen binding fragments of such antibodies. Inaddition, the invention provides cells lines such as isolated cell linesthat produce antibodies of the invention, particularly monoclonalantibodies. As used herein, the term “antibody” is used in its broadestsense to include polyclonal and monoclonal antibodies, as well asantigen binding fragments of such antibodies. With regard to ananti-p/CIP antibody, for example, the term “antigen” means a p/CIPprotein, polypeptide or peptide portion thereof. An anti-p/CIP antibody,or antigen binding fragment of such an antibody, is characterized byhaving specific binding activity for p/CIP or a peptide portion thereofof at least about 1×10⁻⁵ M⁻¹. An anti-p/CIP antibody can have specificbinding activity for p/CIP without binding other NCoA polypeptides suchas NCoA-1 or NCoA-2. Fab, F(ab′)₂, Fd and Fv fragments of an anti-p/CIPantibody, which retain specific binding activity for p/CIP, are includedwithin the definition of an antibody. Similar antibodies can beidentified with respect to the full length murine NCoA-2 polypeptidedisclosed herein.

In addition, the term “antibody” as used herein includes naturallyoccurring antibodies as well as non-naturally occurring antibodies,including, for example, single chain antibodies, chimeric, bifunctionaland humanized antibodies, as well as antigen-binding fragments thereof.Such non-naturally occurring antibodies can be constructed using solidphase peptide synthesis, can be produced recombinantly or can beobtained, for example, by screening combinatorial libraries consistingof variable heavy chains and variable light chains as described by Huseet al., Science 246:1275-1281 (1989), which is incorporated herein byreference. These and other methods of making, for example, chimeric,humanized, CDR-grafted, single chain, and bifunctional antibodies arewell known to those skilled in the art (Winter and Harris, Immunol.Today 14:243-246 (1993); Ward et al., Nature 341:544-546 (1989); Harlowand Lane, Antibodies: A laboratory manual (Cold Spring Harbor LaboratoryPress, 1988); Hilyard et al., Protein Engineering: A practical approach(IRL Press 1992); Borrabeck, Antibody Engineering, 2d ed. (OxfordUniversity Press 1995); each of which is incorporated herein byreference).

Anti-p/CIP antibodies can be raised using as an immunogen asubstantially purified full length p/CIP protein, which can be preparedfrom natural sources or produced recombinantly, or a peptide portion ofa p/CIP polypeptide as defined herein, including synthetic peptides asdescribed above. A non-immunogenic peptide portion of p/CIP can be madeimmunogenic by coupling the hapten to a carrier molecule such bovineserum albumin (BSA) or keyhole limpet hemocyanin (KLH), or by expressingthe peptide portion as a fusion protein. Various other carrier moleculesand methods for coupling a hapten to a carrier molecule are well knownin the art and described, for example, by Harlow and Lane, supra, 1988.

Particularly useful antibodies of the invention are those that bind touncomplexed p/CIP, but not to a p/CIP in a complex, for example, withCBP, and, conversely, those that bind to the complexed form of p/CIP,but not to the uncomplexed form. An anti-p/CIP antibody is useful, forexample, for determining the presence or level of a p/CIP in a tissuesample, which can be a lysate or a histological section. Theidentification of the presence or level of a p/CIP in the sample can bemade using well known immunoassay and immunohistochemical methods(Harlow and Lane, supra, 1988). An anti-p/CIP antibody also can be usedto substantially purify p/CIP from a sample and, in addition, can beused to copurify a protein such as a transcription factor that iscomplexed with the p/CIP polypeptide. An anti-p/CIP antibody can be usedto detect a p/CIP polypeptide in a sample of cells or in an organism.

A kit incorporating an anti-p/CIP antibody, which can be specific forthe complexed or uncomplexed form of p/CIP, can be particularly useful.Such a kit can contain, in addition to an anti-p/CIP antibody, areaction cocktail that provides the proper conditions for performing theassay, control samples that contain known amounts of p/CIP and, ifdesired, a second antibody specific for the anti-p/CIP antibody.

A protein such as an anti-p/CIP antibody, as well as p/CIP or a peptideportion thereof, can be labeled so as to be detectable using methodswell known in the art (Hermanson, “Bioconjugate Techniques” (AcademicPress 1996), which is incorporated herein by reference; Harlow and Lane,supra, 1988; chap. 9). For example, a protein can be labeled withvarious detectable moieties including a radiolabel, an enzyme, biotin,or a fluorochrome or fluorescent moiety, such as a green fluorescentprotein (see U.S. Pat. No. 5,625,048; WO96/23810; WO97/28261;PCT/US97/12410; and PCT/US97/14593, each of which is incorporated hereinby reference). Reagents for labeling a protein such as an anti-p/CIPantibody can be included in a kit containing the protein or can bepurchased separately from a commercial source.

Following contact, for example, of a labeled antibody with a sample suchas a tissue homogenate or a histological section of a tissue,specifically bound labeled antibody can be identified by detecting theparticular moiety. Alternatively, a labeled second antibody can be usedto identify specific binding of an unlabeled anti-p/CIP antibody. Asecond antibody generally will be specific for the particular class ofthe first antibody. Such second antibodies are readily available fromcommercial sources. The second antibody can be labeled using adetectable moiety as described above. When a sample is labeled using asecond antibody, the sample is first contacted with a first antibody,which is an anti-p/CIP antibody, then the sample is contacted with thelabeled second antibody, which specifically binds to the anti-p/CIPantibody and results in a labeled sample.

Methods for raising polyclonal antibodies, for example, in a rabbit,goat, mouse or other mammal, are well known in the art. In addition,monoclonal antibodies can be obtained using methods that are well knownand routine to the skilled person (Harlow and Lane, supra, 1988).Essentially, spleen cells from a p/CIP-immunized mouse can be fused toan appropriate myeloma cell line such as SP/02 myeloma cells to producehybridoma cells. Cloned hybridoma cell lines can be screened usinglabeled p/CIP protein to identify clones that secrete anti-p/CIPmonoclonal antibodies. Hybridomas expressing anti-p/CIP monoclonalantibodies having a desirable specificity and affinity can be isolatedand utilized as a continuous source of the antibodies, which are useful,for example, for preparing standardized kits as described above.Similarly, a recombinant phage that expresses, for example, a singlechain anti-p/CIP antibody also provides a monoclonal antibody that canused for preparing standardized kits.

A monoclonal anti-p/CIP antibody can be used to prepare anti-idiotypicantibodies, which presents an epitope that mimics the epitope recognizedby the monoclonal antibody used to prepare the anti-idiotypicantibodies. Where the epitope recognized by the monoclonal antibodyincludes, for example, an LCD, the anti-idiotypic antibody can act as aninhibitor of p/CIP binding to CBP or p/CIP binding to a transcriptionfactor, thus providing a means to regulate a signal transductionpathway.

The present invention also provides a method of identifying an effectiveagent that alters the association of a p/CIP polypeptide with a secondprotein such as CBP or a transcription factor, or that alters theformation of a complex containing two or three of these proteins. Themethod includes the steps of contacting a p/CIP polypeptide and a secondprotein with an agent under conditions that allow the p/CIP polypeptideto associate with the second protein and detecting an alteredassociation of the p/CIP polypeptide and said second protein. Thealtered association indicates that the agent is an effective agent. In amethod of the invention, the p/CIP polypeptide can have, for example,the amino acid sequence shown in FIG. 1, and the second protein can be,for example, a CBP protein, a nuclear receptor or a CBP/p300-dependenttranscription factor. An altered association can be detected, forexample, by measuring the transcriptional activity of a reporter gene.In a method of the invention, a p/CIP polypeptide can be contacted withan agent in vitro or in a cell, including a prokaryotic cell such as ayeast cell and a eukaryotic cell, such as a mammalian cell, for example,a human cell.

The present invention further provides a method of identifying aneffective agent that alters the association of a NCoA-2 polypeptide witha second protein. The method includes the steps of contacting a NCoA-2polypeptide and a second protein with an agent under conditions thatallow the NCoA-2 polypeptide to associate with the second protein anddetecting an altered association of the NCoA-2 polypeptide and saidsecond protein.

As used herein, the term “second protein” refers to a protein thatspecifically associates with a p/CIP or NCoA-2 polypeptide. It isrecognized, however, that p/CIP and NCoA-2 can associate with more thanone additional protein at the same time to form a complex. Thus, asecond protein is exemplified herein by CBP proteins, by nuclearreceptors and by other CBP/p300-dependent transcription factors, whichform a complex with p/CIP or NCoA-2. Effective agents that alter theassociation, for example, of p/CIP, CBP and a transcription factor canbe extremely valuable in that the agent can modulate transcriptionalactivity of the transcription factor.

The term “agent,” as used herein, means a biological or chemicalcompound such as a simple or complex organic molecule, a peptide, apeptidomimetic, a polypeptide, a nucleic acid, a chemical or a smallmolecule. The screening assays described herein are particularly usefulin that they can be automated, facilitating high through-put screeningof randomly or rationally designed agents or libraries of agents, suchas chemicals, small molecules, drugs, peptides, peptidomimetics orpolypeptides, in order to identify those agents that alter theassociation of p/CIP or NCoA-2 with a second protein. If desired, anagent can be screened individually, or can be screened in combinationwith other agents, for example, in a library.

As used herein, the term “associate” or “association,” when used inreference to a p/CIP or NCoA-2 polypeptide and a second (or second andthird) protein means that the p/CIP or NCoA-2 polypeptide and the secondprotein have a binding affinity for each other such that they form abound complex in vivo or in vitro, including in a cell in culture or ina reaction comprising substantially purified reagents. For convenience,the term “bind” or “interact” is used interchangeably with the term“associate.

The screening assays disclosed herein provide a method of identifying an“effective agent,” which is an agent that can increase or decreased theaffinity of an association between a p/CIP or NCoA-2 polypeptide and asecond protein and that has presumptive therapeutic activity. The term“modulate” or “alter,” as used herein in reference to the association ofa p/CIP or NCoA-2 polypeptide and one or two other proteins, means thatthe affinity of the association is increased or decreased. Effectiveagents that can alter such association and, therefore, complex formationof p/CIP, CBP and a transcription factor, can be useful for modulating asignal transduction pathway and, therefore, expression of genes in thepathway. One skilled in the art understands that an effective agent thatalters the association of p/CIP with a second protein such as a CBPprotein may, additionally alter the association of other proteins withp/CIP. Alternatively, an effective agent can selectively alter theassociation of, for example, p/CIP with a CBP protein without alteringthe association of p/CIP with other proteins.

One skilled in the art understands that an effective agent can functiondirectly or indirectly and by a variety of mechanisms to alter theassociation of a p/CIP polypeptide, or NCoA-2 polypeptide, with a secondprotein. An effective agent can function, for example, as a competitorof the binding interaction between a p/CIP polypeptide and a secondprotein, or between a NCoA-2 polypeptide and a second protein. Forexample, a peptide or peptidomimetic that mimics the structure of theCBP interaction domain or the nuclear receptor interaction domain of ap/CIP polypeptide can be an effective agent that decreases the affinityof the association of a p/CIP polypeptide with a second protein, as canbe a fragment of a CBP protein or nuclear receptor that alters theassociation of p/CIP with a second protein. A peptide portion of p/CIPcomprising an LCD, for example, amino acids 947 to 1084 of p/CIP (seeFIG. 2A) is an example of such an agent, since the peptide inhibits, forexample, retinoic acid-dependent gene activation (see Example I).Additional peptide effective agents, which can be peptides as small asabout five amino acids, can be identified, for example, by screening apeptide library (see, for example, Ladner et al., U.S. Pat. No.5,223,409, which is incorporated herein by reference) using one of theassays described herein.

An effective agent also can bind to a p/CIP or NCoA-2 polypeptide at asite distant from the site of interaction, thereby altering thethree-dimensional conformation of the polypeptide such that the affinityof the association with a second protein is increased or decreased. Aneffective agent also can produce an altered association by promoting amodification such as phosphorylation of a p/CIP or NCoA-2 polypeptide.In addition, an effective agent can sequester or alter the subcellularlocalization of a p/CIP or NCoA-2 polypeptide, thereby modulating theeffective concentration of the polypeptide and the extent to which thepolypeptide can associate with a second protein.

A variety of in vivo and in vitro screening assays for detecting analtered association are well known in the art including, for example,the two hybrid assay, coimmunoprecipitation assays, reporter assays andother well known methods such as equilibrium dialysis. One skilled inthe art understands that methods for distinguishing the specificassociation of a p/CIP, for example, and a second protein from anon-specific interaction are routine and, generally, include performingthe appropriate control experiments to demonstrate the absence ofnon-specific protein binding.

An effective agent can be identified by an altered level of reportergene transcription as compared to a control level of transcription inthe absence of the agent. A particularly useful reporter gene iscytosolic β-lactamase, which can be detected by the CCF2/AM substrate,as described in Tsien et al. (U.S. Pat. No. 5,741,657, which isincorporated herein by reference). If desired, a reporter gene canencode a protein expressed, for example, on the cell surface, and analtered level of reporter gene transcription detected by FACS analysis.

A two-hybrid system, such as the yeast two hybrid system, can beparticularly useful for screening a panel of agents in order to detectan altered association of a p/CIP or NCoA-2 polypeptide with a secondprotein (see Example I). Using a two hybrid system, an effective agentis identified by an altered level of transcription of a reporter gene(see Fields and Song, Nature 340:245-246 (1989), which is incorporatedherein by reference). For example, the level of transcription of areporter gene due to the bridging of a DNA-binding domain p/CIP orNCoA-2 polypeptide hybrid and a transactivation domain-second proteinhybrid can be determined in the absence and presence of an agent.

In some cases, an agent may not be able to cross the yeast cell walland, therefore, cannot enter a yeast cell to alter the association of ap/CIP or NCoA-2 polypeptide with the second protein. The use of yeastspheroplasts, which are yeast cells that lack a cell wall, cancircumvent this problem (Ausubel et al., supra, 1994), which isincorporated herein by reference). In addition, an agent, upon enteringa cell, may require “activation” by a cellular mechanism, which may notbe present in yeast. Activation of an agent can include, for example,metabolic processing of the agent or a modification such asphosphorylation of the agent, which can be necessary to convert theagent into an effective agent. In this case, a mammalian cell line canbe used to screen a panel of agents. A transcription assay such as theyeast two hybrid system described in Example I can be adapted for use inmammalian cells using well known methods (see, for example, Fearon etal., Proc. Natl. Acad. Sci., USA 89:7958-7962 (1992), which isincorporated herein by reference; see, also, Sambrook et al., supra,1989; Ausubel et al., supra, 1994).

An altered association also can be detected using an in vitro screeningassay. In vitro screening assays can utilize, for example, p/CIP or ap/CIP fusion polypeptide such as a histidine-p/CIP fusion protein. Foruse in an in vitro screening assay, the p/CIP or p/CIP fusionpolypeptide should have an affinity for a solid substrate as well as theability to associate with a second protein. Convenient solid substratesinclude columns, beads, filters and other materials well known in theart. If desired, the solid substrate can contain a covalently attachedanti-p/CIP antibody. Alternatively, when a fusion polypeptide such as aHis-p/CIP fusion polypeptide is used in the assay, a nickel chelatesubstrate, which is bound by the histidine component of the fusionprotein, can be used (Invitrogen, Carlsbad, Calif.). Other fusionpolypeptide systems are well known in the art and commerciallyavailable, including glutathione-S-transferase (GST) fusionpolypeptides, which can be immobilized on a glutathione affinity resin(Stratagene, La Jolla, Calif.) or using an anti-GST antibody (DAKO,Carpinteria, Calif.); “FLAG” fusion polypeptides, which can beimmobilized on a substrate using anti-FLAG antibody; “AU” fusionpolypeptides, which can be immobilized on a substrate using anti-AUantibody, commercially available from Berkeley Antibody Co., Richmond,Calif.; or Myc tag fusion polypeptides, which can be immobilized on asubstrate using anti-Myc antibody, commercially available fromInvitrogen. As an alternative to immobilization of the p/CIP or NCoA-2polypeptide, the second protein can be immobilized on a solid substrateusing a fusion protein strategy or antibody, as described above.

An in vitro screening assay can be performed by allowing a p/CIP orNCoA-2 polypeptide or p/CIP or NCoA-2 fusion polypeptide, for example,to bind to a solid substrate, then adding the second protein, and anagent to be tested. Control reactions, which do not contain an agent,can be performed in parallel. Incubation is performed under suitableconditions, which include, for example, an appropriate bufferconcentration, pH, incubation time and temperature. Subsequently, theassociation of the p/CIP or NCoA-2 polypeptide and the second protein inthe absence and presence of an agent can be detected, for example, byattaching a detectable moiety such as a radionuclide, fluorescent orantigenic label to the p/CIP or NCoA-2 polypeptide, and measuring theamount of label that is associated with the solid support. By comparingthe amount of association in the presence of an agent as compared to thecontrol level of association, an effective agent can be identified.

As set forth above, agents to be screened according to a method of theinvention can include a variety of biological or chemical compounds suchas organic molecules, peptides and peptidomimetics, polypeptides ornucleic acids. In particular, such agents to be screened includefragments of p/CIP or NCoA-2 polypeptides and fragments of CBP proteinsor nuclear receptors. Such fragments can be produced by chemical orproteolytic cleavage of the isolated polypeptide. Methods for chemicaland proteolytic cleavage and for purification of the resultantpolypeptide fragments are well known in the art as described above.(See, for example, Deutscher, Methods in Enzymolgy, Vol. 182, “Guide toProtein Purification,” San Diego: Academic Press, Inc. (1990), which isincorporated herein by reference).

A large collection, or library, of chemicals or small molecule drugsalso are agents that can be screened according to a method of theinvention. Polypeptide libraries, random polypeptides, or polypeptidesof interest also are agents that can be screened for activity asdisclosed herein. Polypeptide libraries include, for example, taggedchemical libraries comprising peptides and peptidomimetic molecules.Polypeptide libraries also comprise those generated by phage displaytechnology. Phage display technology includes the expression ofpolypeptide molecules on the surface of phage as well as othermethodologies by which a protein ligand is or can be associated with thenucleic acid which encodes it. Methods for production of phage displaylibraries, including vectors and methods of diversifying the populationof peptides which are expressed, are well known in the art (see, forexample, Smith and Scott, Methods Enzymol. 217:228-257 (1993); Scott andSmith, Science 249:386-390 (1990); and Huse, WO 91/07141 and WO91/07149, each of which is incorporated herein by reference). These orother well known methods can be used to produce a phage display librarywhich can be screened, for example, with one of the disclosed assays toidentify an effective agent that alters the association of a p/CIP orNCoA-2 polypeptide with a second protein.

A peptide portion of p/CIP comprising a helical leucine-rich, chargeddomain (LCD), can inhibit the transcriptional activity of one type ofnuclear receptor, such as the retinoic acid receptor, but not of asecond, related nuclear receptor such as the estrogen receptor, whereasa second LCD of p/CIP can inhibit signal transduction induced byinterferon γ, but not signal transduction induced by retinoic acid.Thus, selected peptide portion of p/CIP or of an NCoA can be useful forselecting regulating gene expression in a cell. Because of the centralnature of p/CIP, NCoA proteins, CBP/p300 and the various transcriptionfactors that form the complexes disclosed herein, an agent such as anLCD peptide can be used to treat various pathologic conditions. Forexample, the transcriptional activity of CBP/p300-dependent proteinsbelonging to the AP-1 and ets families of transcription factors can bespecifically inhibited, thereby providing a means to reduce or preventthe severity of inflammatory diseases or of cancer. In addition,selective activities of nuclear receptors can be potentiated orinhibited, providing beneficial effects in patients suffering frominflammatory disorders, breast cancer or osteoporosis. Also, inhibitionof NF-KB dependent transcription can provide a benefit to patientshaving an inflammatory disorder or atherosclerosis. Thus, an agent thatalters the association of a p/CIP or of NCoA-2 polypeptide with CBP/p300or with a CBP/p300-dependent transcription factor, or alters the abilityof such proteins to form a complex, is useful as a medicament fortreating a pathologic condition.

The ability of nuclear receptor coactivator (NCoA) polypeptides such asp/CIP to interact with nuclear receptors in a ligand-dependent mannerprovides the basis of a method for identifying a ligand of a nuclearreceptor from a library of one or more test agents. If desired, anuclear receptor corepressor (NCoR) can be used in place of a NCoApolypeptide. A method of the invention can be useful for identifying aligand for a nuclear receptor having a previously identified physiologicligand or for identifying a ligand of an orphan receptor having nopreviously identified ligand. Thus, the invention provides a method foridentifying a ligand for a nuclear receptor, including the steps ofcontacting a NCoA polypeptide, or nuclear receptor binding fragmentthereof, and a nuclear receptor with an agent under conditions thatallow the NCoA polypeptide to associate with the nuclear receptor; anddetecting an altered association between the NCoA polypeptide, ornuclear receptor binding fragment thereof, and the nuclear receptor,where an increased association indicates that the agent is an agonisticligand of the nuclear receptor and a decreased association indicatesthat the agent is an antagonistic ligand of the nuclear receptor. Oneskilled in the art understands that the altered association in thepresence of the agent is compared to the association in the absence ofthe agent.

The term “NCoA polypeptide,” as used herein, means a nuclear receptorcoactivator protein that is characterized, in part, as containing one ormore “LXXLL” motifs and by its ability to mediate ligand-dependentnuclear receptor activation. A NCoA polypeptide can be, for example, aSRC-1/NCoA-1, p/CIP or NCoA-2 polypeptide, or a polypeptide havingsubstantial similarity to one of these polypeptides. A NCoA polypeptidecan have, for example, at least about 30% amino acid identity with aSRC-1/NCoA-1, p/CIP or NCoA-2 polypeptide and, further, can have atleast about 40%, 50%, 60%, 70%, 80% or 90% amino acid identity with aSRC-1/NCoA-1, p/CIP or NCoA-2 polypeptide.

The agent to be tested for agonist or antagonist activity can beprovided in purified form, or in impure form as a pool of differentagents. As described above, an agent can be a biological or chemicalcompound such as a simple or complex organic molecule, a peptide, apeptidomimetic, a polypeptide or a nucleic acid. The nuclear receptorcan be, for example, a steroid hormone receptor, retinoid receptor orfatty acid metabolite receptor. Retinoic acid receptors, estrogenreceptors, progesterone receptor and thyroid receptors are examples ofnuclear receptors useful in the claimed methods. The NCoA polypeptidecan be, for example, a p/CIP, NCoA-1 or NCoA-2 polypeptide, or an activefragment of a NCoA polypeptide with nuclear receptor binding activity.Active p/CIP and NCoA-2 polypeptides including a nuclear receptorinteraction domain have been described hereinabove. Such a fragment canbe, for example, a LCD peptide that contains one or more LXXLL motifs.In the presence of an agent that is an agonist, the nuclear receptor canundergo a conformational change, whereby there is an increasedassociation of the nuclear receptor with the NCoA polypeptide or nuclearreceptor interaction domain thereof, or with another coactivatorcontaining one or more LXXLL motifs.

In one embodiment, the nuclear receptor can be immobilized on a solidsubstrate, for example, by expressing the nuclear receptor as a GSTfusion protein and capturing the fusion protein on a glutathioneaffinity matrix. The nuclear receptor fusion protein can be incubated,for example, with labeled NCoA polypeptide such as labeled p/CIPpolypeptide in the presence of an agent to be tested. Followingincubation and subsequent washing of the glutathione affinity matrix,specifically bound NCoA polypeptide can be detected quantitatively,semi-quantitatively, or qualitatively. In the presence of a ligand thatis an agonist, the association of the p/CIP or other NCoA polypeptidewith the GST matrix is increased. Conversely, in the presence of aligand that is an antagonist of the nuclear receptor, the association ofthe NCoA polypeptide with the GST matrix is decreased.

As set forth above, a NCoA polypeptide such as p/CIP can be labeled witha variety of labels including fluorescent labels or radiolabels such as³⁵S-labeled amino acids, which can be incorporated by in vitrotranslation using a rabbit reticulocyte translation system. A LCDpeptide containing at least one LXXLL motif also can be used, ifdesired, in place of or in addition to, the NCoA polypeptide. Such a LCDpeptide can be modified to contain N- or C-terminal tyrosine residuesthat do not substantially influence interaction with the nuclearreceptor but which can be conveniently labeled, for example, usingradioiodination. A short sequence tag suitable for phosphorylation of anLCD peptide with ³²P-ATP also can be used as a label. Fluorescentdetection, for example using green fluorescent protein, can beparticularly useful in the methods of the invention. Useful fluorescentdetection methods include florescence polarization as well asfluorescence resonance energy transfer (FRET)-based assays. FRET-basedassays are particularly advantageous for high throughput screeningapproaches since such assays are homogeneous and do not require awashing step and, in addition, can be useful for detecting nuclearreceptor interactions within a cell.

In the methods of the invention, the ligand to be identified can be anantagonist. The present invention provides, for example, a method ofidentifying an antagonist of a nuclear receptor. The method includes thesteps of contacting a NCoA polypeptide or nuclear receptor interactiondomain thereof and a nuclear receptor with an agonist of the nuclearreceptor and an agent under conditions that allow the NCoA polypeptideor nuclear receptor interaction domain thereof to associate with saidnuclear receptor; and detecting an altered association of the NCoApolypeptide or nuclear receptor interaction domain thereof and thenuclear receptor, where a decreased association indicates that the agentis an antagonist of the nuclear receptor. An agent that is an antagonistcan compete with the agonist for binding to the nuclear receptor withoutinducing the conformation change required for interaction of the NCoApolypeptide, or nuclear receptor interaction domain thereof, and thenuclear receptor. As described above, a LCD peptide containing one ormore LXXLL motifs can be substituted for a NCoA polypeptide in themethods of the invention.

In a further embodiment, the invention provides a method of identifyinga ligand with mixed agonist and antagonist properties with respect to aparticular nuclear receptor. Such a method includes the steps of:contacting a first NCoA polypeptide, or nuclear receptor interactiondomain thereof, and a nuclear receptor with an agent to form a firstcomplex; detecting an altered association of the first complex in thepresence and absence of the agent; contacting a second NCoA polypeptide,or nuclear receptor interaction domain thereof, and a nuclear receptorwith the agent to form a second complex; and detecting an alteredassociation of the second complex in the presence and absence of theagent, wherein an increased association of the first complex combinedwith a decreased association of the second complex or a decreasedassociation of the first complex combined with an increased associationof the second complex indicates that the agent is a ligand of thenuclear receptor having mixed agonist and antagonist activity. A ligandidentified by this method can be particularly useful since it canexhibit different effects on nuclear receptor function in different celltypes and can be useful for differentially modulating different classesof transcription factors.

The following example is intended to illustrate but not limit thepresent invention.

EXAMPLE I Identification and Characterization of p/CIP

This Example provides methods for isolating and characterizing thenucleic acid molecule encoding p/CIP, which regulates the activity ofCBP/p300-dependent transcription factors. Additional details related tothese methods are provided in Torchia et al., June 1997 (Nature), whichis incorporated herein by reference.

A. Materials and Methods

1. Isolation of Interacting Proteins

Expression cloning was performed, using a ³²P-labeled GST-CBP(2058-2170) or ³²P-labeled GST-ER ligand binding domain probe in thepresence of 10⁻⁶ M estradiol (Kamei et al., Cell 85:1-12 (1996), whichis incorporated herein by reference). cDNA's corresponding to p/CIP,NCoA-1 and NCoA-2 were assembled into PCMX and tested by in vitrotranslation, generating products which all migrated at approximately 160kDa. Databank accession numbers for p/CIP and NCoA-2 sequences areAF000581 (p/CIP) and AF000582 (NCoA-2), each of which is incorporatedherein by reference.

2. Yeast Two-hybrid Interaction Assays

The yeast strain EGY 48, the LexA-β-galactosidase reporter construct(PSH 18-34) and the B42 parental vectors (pEG 202 and pJG 4-5) were allpreviously described (Gyuris et al., Cell 75:791-803 (1993), which isincorporated herein by reference; Kamei et al., supra, 1996). Nuclearreceptor ligand binding domains and various CBP fragments were subclonedinto PEG 202 bait vector. DNA fragments encompassing the entirep/CIP-NCoA-1 or NCoA-2 proteins were generated either by using anappropriate restriction digest or by PCR and subcloned into pJG 4-5 preyvectors. EGY 48 cells were transformed with the lac Z reporter plasmidpSH 18-34 with the appropriate bait and prey vectors and plated out on-Ura-His-Trp medium containing 2% galactose. Isolated yeast colonieswere allowed to grow in the same liquid medium, followed by assaying forβ galactosidase, as previously described (Ausubel et al., supra, 1994).

3. Transient Transfections and Reporter Assays

Transfection experiments were conducted in either HELA or CV-1 cellsusing the standard calcium phosphate procedure. Typically, 1 ug of aRARE- or ERE-driven luciferase reporter were cotransfected with 1 ug ofthe indicated vectors. The final DNA concentration was adjusted to 10ug/60 mm dish, incubated for 24 hr, then the appropriate ligands wereadministered for 24 hr at a concentration of 10⁻⁶ M. Alternatively,cotransfection experiments were conducted using a PCMX p/CIP, NCoA-1 orPCR-generated NCoA-1 fragments fused to the GAL 4 DNA binding domain (aa1-147). Cells were transfected with 1 ug of a (UAS)₆-luciferase reporterand the indicated concentrations of GAL4 fusion proteins, then harvested48 hrs later.

4. Affinity Purified NCoA Antibodies and Peptides

cDNA fragments corresponding to p/CIP (544-851), NCoA-1 (424-789) orNCoA-2 (787-1129) were subcloned into the pM vector containing anin-frame His tag and recombinant His-tagged proteins were generated andpurified by nickel chelate chromatography. The purified recombinantproteins were injected into rabbits and antibodies were generated andaffinity purified using standard procedures (Harlow and Lane, supra,1988. Peptide sequences were generated (Research Genetics) and confirmedby mass spectrosopy analysis, including NCoA-1 LCD1 (aa 631-647); NCoA-1LCD2 (687-706); NCoA-1 LCD4 (aa 907-926); CBP N′-PL (aa 1-19); and CBPN′-P2 (aa 8-19).

5. Interaction Assays, Immunoprecipitations and Enzymatic Assays

Whole cell extracts were prepared by lysing the cells in NET-N buffercontaining 50 mM Tris (pH 7.6), 5 mM EDTA, 0.3 M NaCl, 1 mM DTT, 0.1%NP-40 and protease inhibitors (0.2 mM PMSF, 10 ug/ml each of leupeptin,pepstatin and aprotinin), centrifuged at 30,000×g for 1 hr at 4° C. andthe supernatant was stored at −80° C. until use.

GST-RAR (143-462), GST-ER(251-595) and GST-CBP(2058-2170) were generatedas described (Kamei et al., supra, 1996). 25 ul of GST SEPHAROSE beadscontaining 10 ug of the GST recombinant proteins were incubated in thepresence or absence of the appropriate ligand for 30 min at roomtemperature, followed by the addition of 1 mg of cell extract andincubated for an additional 1 hr at 4° C. The complexes were thencentrifuged, washed three times in NET-N buffer, separated by SDS-PAGEand western blotted with the appropriate antibodies (1 ug/ml). Forco-immunoprecipitation assays, 1 mg of cell extract was incubated in thepresence of 2 ug of p/CIP or NCoA antibody for 2 hr at 4° C. The immunecomplexes were precipitated with protein A SEPHAROSE (50% w/v). Proteincomplexes were separated by SDS-PAGE (Laemmle, Nature 227:680-685(1970), which is incorporated herein by reference) and western blottedusing 1 ug/ml of an anti-CBP/P300 monoclonal antibody (UBI). For invitro competition assays, the indicated peptides were incubated with invitro translated NCoA-1 prior to GST interaction with RAR.

6. Mutagenesis

Mutations in NCoA-1 and CBP were introduced by site-directed mutagenesisusing the quick change mutagenesis kit according to the manufacturersinstructions (Stratagene; La Jolla Calif.). Double strandedoligonucleotides were designed such that the wild type sequencecorresponding to amino acids 695 to 698 and amino acids 756 to 759 inpCMX NCoA-1 and pJG4-5-4 NCoA-1(aa 635-760) were substituted withalanines. A similar protocol was used to replace amino acids 70 to 73 inPJG4-5 CBP(aa 1-101).

7. Single Cell Microinjection Assay

Insulin-responsive Rat-1 fibroblasts were seeded on acid washed glasscoverslips at subconfluent density and grown in MNE/F12 mediumsupplemented with 10% fetal bovine serum, gentamicin and methotrexate.Prior to the injection, the cells were rendered quiescent by incubationin serum-free medium for 24-36 hr. Plasmids were injected into thenuclei of cells at a final concentration of 100 mg/ml. Peptides wereinjected at a concentration of 200 mM. Either preimmune IgG of theappropriate species or antibodies directed against p/CIP, NCoA-1 orNCoA-2 were co-injected and allowed the unambiguous identification ofthe injected cells.

Microinjections were carried out using an Eppendorf semiautomatedmicroinjection system mounted on an inverted Zeiss microscope.Approximately 1 hr after injection, the cells were stimulated, whereindicated, with the appropriate ligand. In the case of rescueexperiments, the cells were stimulated with ligand 6 hr after injection,to allow protein expression. After overnight incubation, the cells werefixed, then stained to detect injected IgG and β-galactosidaseexpression (Rose et al., J. Cell. Biol. 119:1405-1411 (1992), which isincorporated herein by reference; Kamei et al., supra, 1996). Injectedcells were identified by staining with tetramethylrhodamine-conjugateddonkey anti-rabbit IgG.

B. Results

1. Identification of Novel Members of the Nuclear Receptor Co-ActivatorFamily

The initial expression screening strategy for identifying members of thep160 gene family was based on the observation that thebiochemically-identified p160 proteins interacted with a 100 amino acidregion in the C-termini of CBP (aa 2058-2170), as well as the ligandedestrogen receptor (ER; Ogryzko et al., Cell 87:953-960 (1996), which isincorporated herein by reference). This strategy allowed isolation ofthe previously reported NCoA-1/SRC-1 protein and of a second relatedfactor, NCoA-2 (FIG. 2a), which has a molecular mass of 159.6 kDa andappears to be the murine homologue of the human TIF-2, a portion ofwhich has been recently reported as GRIP-1 (Voegel et al., EMBO J.15(14):3667-3675 (1996); Hong et al., Proc Natl. Acad. Sci. USA93:4948-4952 (1996), each of which is incorporated herein by reference).In addition, a related factor was identified and is designated herein asp300/CBP/Co-Integrator-Associated Protein.

p/CIP is a 152 kDa protein that is highly related to SRC-1/NCoA-1 andNCoA-2/TIF-2, showing an overall amino acid identity of 31% and 36%,respectively (FIG. 2a). p/CIP has a conserved N-terminal bHLH, PAS “A”domain (50-60% amino acid identity), a serine/threonine rich region, anda C-terminal glutamine-rich region, each of which also is present inNCoA-1 and NCoA-2. Western blot analysis indicates that p/CIP, NCoA-1and NCoA-2 are widely expressed in adult tissues and in all cell linesevaluated (FIGS. 2b and 2 c).

2. A CBP/pCIP Complex

To evaluate the association of p/CIP, NCoA-1, and NCoA-2 with CBP andnuclear receptors, GST-CBP(2058-2170) was used to affinity purifyinteracting proteins from HeLa cell extracts. p/CIP was consistentlyobserved by immunoblotting using affinity purified anti-p/CIP IgG,whereas much smaller amounts of NCoA-1 were detected followingimmunoblotting with anti NCoA-1 IgG (FIG. 3a). Similarly,immunoprecipitations from whole cell extracts using excess antiseraselective for each protein, followed by immunoblotting withanti-CBP/p300 antibody, demonstrated that the vast majority of CBP/p300coprecipitated with p/CIP, although small amounts of NCoA-1- andNCoA-2-associated CBP were detected (FIG. 3b). Conversely, the amount ofCBP/p300 remaining in the supernatant fraction following immunodepletionwith anti-NCoA-1 IgG remained unchanged, while a significant fraction ofCBP was removed following immunodepletion with anti-p/CIP IgG (FIG. 3b).These results indicate that p/CIP forms a complex with CBP in the cell.

To further define the CBP interaction domain in p/CIP, deletion mutantswere generated and tested against CBP(2058-2170) using a yeasttwo-hybrid assay. The major CBP interaction domain was located betweenamino acids 758 to 1115 of p/CIP, with an internal 200 amino acid domainstill capable of interacting. Interestingly, a less pronouncedinteraction was observed with the N-terminal region containing the PAS“A” domain (FIG. 3c). A single nuclear receptor interaction domain (aa591-803) was localized N-terminal of the CBP/p300 interaction domain(FIG. 3c). Further mapping delineated a minimal nuclear receptorinteraction region encompassing amino acids 680-740 in p/CIP that wassufficient for binding to the liganded nuclear receptors. Comparableregions in NCoA-1 and NCoA-2 were found to mediate interactions withboth CBP/p300 and nuclear receptors (FIG. 3d). GST pull-down assays ofwhole cell extracts revealed that p/CIP, NCoA-1, and NCoA-2 interactedwith GST-ER and GST-RAR in a ligand-dependent manner (FIG. 3e).

Cotransfection with NCoA-1/SRC-1 or NCoA-2/TIF-2 expression vectorsclearly potentiated ligand-dependent activation events (generally 3-foldto 8-fold), while cotransfection with p/CIP expression plasmids resultedin minimal or no activation effects (FIG. 3f, left). In addition, whenfull length cDNA's were fused to GAL4(1-147), the activation observed byGAL-NCoA-1 was significantly stronger than GAL-p/CIP (FIG. 3f, right).Cotransfection of CBP and NCoA-1 or NCoA-2 expression vectors resultedin variable synergy (data not shown), consistent with previous findingsreported for SRC-1 (Smith et al., Proc. Natl. Acad. Sci., USA93:8884-8888 (1996)).

To investigate the functional roles of p/CIP, NCoA-1 and NCoA-2,microinjection studies were performed, using the affinity-purifiedIgG's. Reporter genes were placed under the control of a minimalpromoter containing either nuclear receptor or other response elements(Kamei et al., supra, 1996). Microinjection of anti-p/CIP IgG eliminatedthe ability of retinoic acid to activate an RAR-dependent transcriptionunit (FIG. 4a), but was without effect on a promoter under the controlof SP-1 elements or the CMV promoter. In similar experiments, p/CIP alsowas required for the actions of estrogen, thyroid hormone andprogesterone receptors (FIG. 4b).

To determine whether depletion of CBP, rather than p/CIP itself, wasresponsible for the observed effects, the relative abilities of p/CIP,CBP, NCoA-1, and/or NCoA-2 to rescue the inhibitory effect of anti-p/CIPIgG was evaluated. No factor alone, including CBP, was able to rescuethe inhibitory effect of anti-p/CIP IgG on RAR-dependent transcription,indicating that steric blockage or removal of CBP did not account forthe observed effects. However, the simultaneous expression of both p/CIPand CBP fully restored retinoic acid transcriptional response inanti-p/CIP-treated cells (FIG. 4c). These results indicate that both CBPand p/CIP are required together for nuclear receptor activation.

To independently confirm the need for p/CIP, the effect of a 137 aminoacid region of p/CIP (aa 947-1084) containing the core CBP interactiondomain was tested by microinjection assays. This peptide completelyinhibited retinoic acid-dependent gene activation (FIG. 4d; left), butdid not block the activity of non-CBP-dependent promoters (FIG. 4d;right).

The requirement of p/CIP for transcriptional activation by otherCBP-dependent transcription factors, such as STAT also was examined(Bhattacharya et al., Nature 383:344-347 (1996); Zhang et al., Proc.Natl. Acad. Sci., USA 93:15092-15096 (1996); Horvai et al., Proc. Natl.Acad. Sci,. USA 94:1074-1079 (1997)). The effect of anti-p/CIP andNCoA-1 IgG was evaluated by immunoinjection assay in cells, initiallyusing interferon γ-dependent or TPA-dependent reporters. Anti-p/CIP IgGentirely inhibited the STAT-dependent and TPA-dependent transcriptionalactivation events (see FIG. 4e) and the inhibition was not restored byoverexpression of CBP, alone. Independent confirmation was provided byover-expression of the CBP interaction domain of p/CIP (aa 947-1084),which blocked the ability of interferon γ or of TPA to stimulatetranscriptional activation (FIG. 4e). Further, C-terminally truncatedCBP failed to enhance either interferon γ- or TPA-dependenttranscription in cotransfection assays and could not rescue the block ofretinoic acid- and interferon γ-dependent gene activation by injectedanti-CBP IgG (Kamei et al., supra, 1996; Horvai et al., supra, 1997).These results indicate that p/CIP and CBP represent a functionalcomplex, required for function by several CBP-dependent transcriptionfactors in addition to nuclear receptors.

3. Roles of NCoA-1 and NCoA-2 in Nuclear Receptor Activation

Based on the requirement for p/CIP in both nuclear receptor and severalCBP/p300-dependent transcription factors, it was important to evaluatethe precise roles of NCoA-1/SRC-1 and NCoA-2/TIP-2, which, bycotransfection, enhance transactivation by nuclear receptors.Microinjection of anti-NlCoA-1 IgG, but not of anti-NCoA-2 IgG,effectively inhibited retinoic acid-dependent transcription (FIG. 5a),while these antisera failed to inhibit several control promoters thatlacked nuclear receptor response elements (FIG. 5a). In addition,anti-NCoA-1 IgG fully inhibited estrogen and thyroid hormone receptorstimulation (FIG. 5b) and partially inhibited progesterone receptorstimulation (FIG. 5b). Co-injection of NCoA-1, NCoA-2 or p/CIPexpression vectors revealed that the inhibitory effects of anti-NCoA-1IgG could be entirely reversed by either NCoA-1 or NCoA-2, but not byp/CIP (FIG. 5c) consistent with a distinct role for this factor, and incontrast to the requirement for both p/CIP and CBP to rescue theinhibitory actions of anti-p/CIP IgG. Co-injection of a CMV-CBPexpression vector also failed to restore activity, consistent with themodel that both NCoA-1 and the CBP/p300/p/CIP complex are independentlyrequired for nuclear receptor gene activation events (FIG. 5c). Incontrast, anti-NCoA-1 IgG exerted no effects on either cAMP- orinterferon γ-dependent reporters (FIG. 4f). These results indicate thatNCoA-1 is selectively required as a coactivator for the ligand-activatednuclear receptor gene expression events; the requirement for theCBP/p300/p/CIP complex reflects a more general obligatory role in geneactivation events.

4. Interaction Motifs of the Co-Integrator Complex Selectively InhibitTranscrintional Effects of Distinct Signal Transactivation Pathways

In view of the relatedness of NCoA-1 and p/CIP, despite the apparentdistinctions in their functional roles, and the involvement of CBP/p300in activation of different classes of transcription factors, the abilityof distinct interaction domains to selectively block the actions ofspecific signal transduction pathways at a nuclear level was examined.Delineation of the nuclear receptor interaction domains of p/CIP,NCoA-1, and NCoA-2 revealed the presence of highly conserved leucine,charged residue-rich domains (LCD's) that share a consensus coresequence, LXXLL (FIG. 6a). This motif is found in both the nuclearreceptor and the p/CIP interaction domains of CBP and in the CBPinteraction domain of p/CIP.

Analysis of these putative interaction regions by the self-optimizedprediction method (SOPM; Geourjon and Deleage, Protein Engineering7:157-164 (1994), which is incorporated herein by reference) stronglysuggested that they represent helical domains, generally withamphipathic characteristics (FIG. 6a). To begin to investigate whetherthese LCD's exert a critical interaction function, four amino acidmutations of this motif were introduced into the N-terminus of CBP (aa65-76), abolishing interactions with nuclear receptors (FIG. 6b). Theminimal nuclear receptor interaction domain of NCoA-1 contains threesuch helical motifs, and a fourth such motif (LCD6) also is present in avariant of NCoA-1 (Onate et al., Science 270:1354-1357 (1995); Kamei etal., supra, 1997).

“To assess the importance of these motifs in NcoA-1, a smaller regionlacking helical domain 3 resulted in little or no decrease in binding toeither estrogen or retinoic acid receptors, while deletion of helicaldomain 1 exhibited a small but significant decrease (FIG. 6c). Incontrast, a four amino acid substitution in the second NCoA-1 helicaldomain (LCD2; HRLL (SEQ ID 5, aa 10-13)→AAAA (SEQ ID 13, aa 3-6)), whichwould alter the properties of this helix, abolished interaction withboth estrogen and retinoic acid receptors. Conversely, a 37 amino acidregion of NCoA-1 containing LCD2, or 59 amino acids containing LCD6, wassufficient for binding to liganded nuclear receptors (FIG. 6c, left).The addition of an excess 24 mer oligopeptide encompassing LCD2effectively blocked interactions between liganded RAR and NcoA-1 invitro; whereas a peptide corresponding to LCD1 was less effective. Theseresults indicate that specific motifs can be both required and, incertain instances, sufficient, for interaction.”

To assess the potential selective functional requirements of thesehelical motif sequences in the nuclear receptor interaction domain ofNCoA-1, mutations in helical domains 2 or 3 were generated in thecontext of the holoprotein and tested for the ability to rescueanti-NCoA-1 IgG inhibition of retinoic acid receptor function. Whereaswild-type NCoA-1 fully rescued activation, an NCoA-1 holoproteinharboring clustered point mutations in helical domain 3 (LCD3-mut) wascompletely ineffective at rescuing retinoic acid receptor function.NCoA-1 containing a helical domain 2 (LCD2-mut) mutation retained someresidual efficacy (FIG. 6d), consistent with the residual ability of thehelical domains to mediate nuclear receptor interactions. Surprisingly,however, NCoA-1 harboring the helical domain 3 mutation (LCD3-mut)retained full functional ability in estrogen receptor-dependent geneactivation, whereas LCD2-mut was completely ineffective at rescuingestrogen receptor function (FIG. 6e). These results indicate that thehelical interaction motifs of NCoA-1 afford a level of receptorspecificity.

To independently assess the importance of these motifs, correspondingpeptides were tested for their ability to inhibit specific activationevents. NCoA-1 harbors two additional related helical interactionmotifs, and a peptide encompassing one of these motifs (LCD4) can blocknuclear receptor transcription factor function and does not impair STATfunction (FIG. 7a). Furthermore, a mutation within this motif markedlyimpairs the function of this region of p/CIP (data not shown). Thus,specific signal transduction pathways can be selectively blocked bydistinct helical interaction motifs.

Other motifs, not required for nuclear receptor activation, also wereexamined to determine if, similarly, they are critical for coactivatorfunction for other classes of CBP-dependent transcription factors, thusproviding a means to selective block distinct signal transductionpathways. This study was initiated based on the demonstration that acritical STAT interaction domain is found within the first 100 aminoacids of CBP by coimmunoprecipitation (Horvai et al., supra, 1997). Todetermine whether a sequence of the CBP N-terminal 100 amino acids,distinct from the nuclear receptor motif, can both mediate interactionswith STAT-1 and be required for STAT function, the effects of peptidescorresponding to N-terminal regions of CBP on STAT-1 or retinoic acidreceptor function was evaluated. Remarkably, a synthetic peptide againstthe N-terminal 22 amino acids of CBP (CBP N′P1; FIG. 7b) markedlyinhibited interferon γ-dependent gene activation, but was without effecton retinoic acid receptor function. The identical peptide, from whichthe N-terminal seven amino acids (MAENLLY) were deleted, abolished thiseffect (CBP N′-P2; FIG. 7b), indicating that this sequence encompassed amotif required for STAT interaction and function. These results furthersupport the functional significance of the STAT-1 interaction motifpreviously identified in the CBP N-terminus (Horvai et al., supra,1997).

In parallel, the ability of the CBP N-terminal peptide to selectivelyblock the inhibitory effects of STAT-1 or retinoic acidreceptor-dependent transcription was examined by evaluating its effectson simultaneous stimulation by interferon γ and retinoic acid. Thesimultaneous addition of retinoic acid and interferon γ led toreciprocal inhibition of retinoic acid- and interferon-dependentreporter gene expression (FIG. 7c). However, the addition of the CBPN-terminal 22 amino acid peptide (CBP N′-P1) relieved inhibition ofRAR-dependent transcription by interferon γ, consistent with thehypothesis that this inhibitory effect represents, at least in part,competition for CBP coactivator complexes, analogous to that proposedfor AP-1 and nuclear receptors (Kamei et al., supra, 1997). Together,these results are consistent with the hypothesis that different motifsare used in assembling CBP-dependent complexes by different classes oftranscription factors, and that peptides based on these motifs canselectively block specific signal transduction pathways.

The results disclosed herein indicate that p/CIP, which is associatedwith CBP/p300 in cell, is involved in regulating transcription bynuclear receptors and by other CBP-dependent factors, including STAT andAP-1. Furthermore, both the CBP/p/CIP complex and NCoA-1 are required topermit full ligand-activated gene transcription in the cells examined,while NCoA-1/SRC-1 is not required for other CBP-dependenttranscription. Because CBP is capable of associating with a large numberof additional factors, including myb, YY1, SREBP, myoD and the HLH1factors, it is likely that p/CIP and CBP are components of a largercomplex important for integration of many signal transduction pathways.

Studies have shown that the N-terminus of CBP alone is sufficient topotentiate CREB function using transient cotransfection assays (Bisottoet al., J. Biol. Chem. 271:17746-17750 (1996); Swope et al., J. Biol.Chem. 271:28138-28145 (1996)). In contrast, a recent study has shownthat the C-terminus also was required in in vitro transcription assays(Nakajima et al., Genes and Devel. 11:738-747 (1997)). While not wishingto be bound by the following, the results disclosed herein indicate thatconformational alterations in CBP holoprotein, which may be contributedby p/CIP, can modulate interactions with transcription factors andassociated regulatory proteins, including protein kinases and those thathave been shown to possess histone acetylase functions. Furthermore,p/CAF is capable of interacting with NCoA-1 as well as CBP41, althoughits role in mediating the transcriptional activation by nuclearreceptors is unclear.

The nuclear receptor and CBP interaction domains within NCoA-1, NCoA-2,and p/CIP contain putative helical domains, referred to as LCD's, thatare required and, in at least in some cases, sufficient, forreceptor-specific interactions. Thus, the third helical domain in thenuclear receptor interaction domain of NCoA-1 is differentiallyutilized, being important for retinoic acid function, but not forestrogen receptor-dependent gene activation events. Similar LCD's arepresent in CBP and in other factors, including TIF-1 and RIP 140, aswell as in the N-terminal interaction domain of p/CAF. Thus, manyfactors can have the ability to associate with the complexes formed onreceptor homodimers or heterodimers bound to their cognate DNA site andcontribute to the specificity of nuclear receptor pathways. Such anassembly of specific complexes of proteins based on these interactionmotifs can provide a basis for receptor-specific and regulated aspectsof nuclear receptor function.

As disclosed herein, helical interaction domains in CBP/p/CIP/NCoAproteins and other nuclear receptor interacting factors permitted theuse of such domains to selectively block gene activation events inresponse to specific signal transduction pathways. Thus, peptidescorresponding to CBP interaction domains selectively block nuclearreceptor or STAT-1 function. The actions of specific inhibitory peptidesindicates that partitioning of CBP accounts, at least in part, fortrans-repression of nuclear receptor, STAT and AP-1 pathways.

14 1 4860 DNA Mus musculus misc_feature (3121)..(3121) “n” is anynucleotide 1 ggcggcgaac ggatcaaaag aatttgctga acagtggact ccgagatcggtaaaacgaac 60 tcttccctgc ccttcctgaa cagctgtcag ttgctgatct gtgatcagg atgagt gga 118 Met Ser Gly 1 cta ggc gaa agc tct ttg gat ccg ctg gcc gctgag tct cgg aaa cgc 166 Leu Gly Glu Ser Ser Leu Asp Pro Leu Ala Ala GluSer Arg Lys Arg 5 10 15 aaa ctg ccc tgt gat gcc cca gga cag ggg ctt gtctac agt ggt gag 214 Lys Leu Pro Cys Asp Ala Pro Gly Gln Gly Leu Val TyrSer Gly Glu 20 25 30 35 aag tgg cga cgg gag cag gag agc aag tac ata gaggag ctg gca gag 262 Lys Trp Arg Arg Glu Gln Glu Ser Lys Tyr Ile Glu GluLeu Ala Glu 40 45 50 ctc atc tct gca aat ctc agc gac atc gac aac ttc aatgtc aag cca 310 Leu Ile Ser Ala Asn Leu Ser Asp Ile Asp Asn Phe Asn ValLys Pro 55 60 65 gat aaa tgt gcc atc cta aag gag aca gtg aga cag ata cggcaa ata 358 Asp Lys Cys Ala Ile Leu Lys Glu Thr Val Arg Gln Ile Arg GlnIle 70 75 80 aaa gaa caa gga aaa act att tcc agt gat gat gat gtt caa aaagct 406 Lys Glu Gln Gly Lys Thr Ile Ser Ser Asp Asp Asp Val Gln Lys Ala85 90 95 gat gtg tct tct aca ggg cag gga gtc att gat aaa gac tct tta gga454 Asp Val Ser Ser Thr Gly Gln Gly Val Ile Asp Lys Asp Ser Leu Gly 100105 110 115 ccg ctt tta cta cag gca ctg gat ggt ttc ctg ttt gtg gtg aatcga 502 Pro Leu Leu Leu Gln Ala Leu Asp Gly Phe Leu Phe Val Val Asn Arg120 125 130 gat gga aac att gta ttc gtg tca gaa aat gtc aca cag tat ctgcag 550 Asp Gly Asn Ile Val Phe Val Ser Glu Asn Val Thr Gln Tyr Leu Gln135 140 145 tac aag cag gag gac ctg gtt aac aca agt gtc tac agc atc ttacat 598 Tyr Lys Gln Glu Asp Leu Val Asn Thr Ser Val Tyr Ser Ile Leu His150 155 160 gag cca aga cgg aag gat ttc tta aac act tac caa aat cca cagtta 646 Glu Pro Arg Arg Lys Asp Phe Leu Asn Thr Tyr Gln Asn Pro Gln Leu165 170 175 atg gag ttt ctt gga cta atg aga acc aga gac aaa aaa gcc ccatac 694 Met Glu Phe Leu Gly Leu Met Arg Thr Arg Asp Lys Lys Ala Pro Tyr180 185 190 195 att tta att gtc cgt atg ttg atg aaa aca cac gac att ttggaa gac 742 Ile Leu Ile Val Arg Met Leu Met Lys Thr His Asp Ile Leu GluAsp 200 205 210 gtg aat gcc agt ccc gaa acg cgc cag aga tat gaa aca atgcag tgc 790 Val Asn Ala Ser Pro Glu Thr Arg Gln Arg Tyr Glu Thr Met GlnCys 215 220 225 ttt gcc ctg tct cag cct cgc gct atg ctg gaa gaa gga gaagac ttg 838 Phe Ala Leu Ser Gln Pro Arg Ala Met Leu Glu Glu Gly Glu AspLeu 230 235 240 cag tgc tgt atg atc tgc gtg gct cgc cgc gtg act gcg ccattc cca 886 Gln Cys Cys Met Ile Cys Val Ala Arg Arg Val Thr Ala Pro PhePro 245 250 255 tcc agt ccc gag agc ttt att acc aga cat gac ctt tcc ggaaag gtt 934 Ser Ser Pro Glu Ser Phe Ile Thr Arg His Asp Leu Ser Gly LysVal 260 265 270 275 gtc aat ata gat aca aac tca ctt aga tct tcc atg aggcct ggc ttt 982 Val Asn Ile Asp Thr Asn Ser Leu Arg Ser Ser Met Arg ProGly Phe 280 285 290 gaa gac ata atc cga aga tgt atc cag agg ttc ttc agtctg aat gat 1030 Glu Asp Ile Ile Arg Arg Cys Ile Gln Arg Phe Phe Ser LeuAsn Asp 295 300 305 ggg cag tca tgg tcc cag aag cgt cac tat caa gaa gcttat gtt cat 1078 Gly Gln Ser Trp Ser Gln Lys Arg His Tyr Gln Glu Ala TyrVal His 310 315 320 ggc cac gca gag acc ccc gtg tat cgt ttc tcc ttg gctgat gga act 1126 Gly His Ala Glu Thr Pro Val Tyr Arg Phe Ser Leu Ala AspGly Thr 325 330 335 att gtg agt gcg cag aca aaa agc aaa ctc ttc cgc aatcct gta acg 1174 Ile Val Ser Ala Gln Thr Lys Ser Lys Leu Phe Arg Asn ProVal Thr 340 345 350 355 aat gat cgt cac ggc ttc atc tcg acc cac ttt cttcag aga gaa cag 1222 Asn Asp Arg His Gly Phe Ile Ser Thr His Phe Leu GlnArg Glu Gln 360 365 370 aat gga tac aga cca aac cca atc ccg cag gac aaaggc atc cga cct 1270 Asn Gly Tyr Arg Pro Asn Pro Ile Pro Gln Asp Lys GlyIle Arg Pro 375 380 385 cct gca gca ggg tgt ggc gtg agc atg tct cca aatcag aat gta cag 1318 Pro Ala Ala Gly Cys Gly Val Ser Met Ser Pro Asn GlnAsn Val Gln 390 395 400 atg atg ggc agc cgg acc tat ggc gtg cca gac cccagc aac aca ggg 1366 Met Met Gly Ser Arg Thr Tyr Gly Val Pro Asp Pro SerAsn Thr Gly 405 410 415 cag atg ggt gga gct agg tac ggg gct tct agt agcgta gcc tca ctg 1414 Gln Met Gly Gly Ala Arg Tyr Gly Ala Ser Ser Ser ValAla Ser Leu 420 425 430 435 acg cca gga caa agc cta cag tcg cca tct tcctat cag aac agc agc 1462 Thr Pro Gly Gln Ser Leu Gln Ser Pro Ser Ser TyrGln Asn Ser Ser 440 445 450 tat ggg ctc agc atg agc agt ccc ccc cac ggcagt cct ggt ctt ggc 1510 Tyr Gly Leu Ser Met Ser Ser Pro Pro His Gly SerPro Gly Leu Gly 455 460 465 ccc aac cag cag aac atc atg att tcc cct cggaat cgt ggc agc cca 1558 Pro Asn Gln Gln Asn Ile Met Ile Ser Pro Arg AsnArg Gly Ser Pro 470 475 480 aag atg gcc tcc cac cag ttc tct cct gct gcaggt gca cac tca ccc 1606 Lys Met Ala Ser His Gln Phe Ser Pro Ala Ala GlyAla His Ser Pro 485 490 495 atg gga cct tct ggc aac aca ggg agc cac agcttt tct agc agc tcc 1654 Met Gly Pro Ser Gly Asn Thr Gly Ser His Ser PheSer Ser Ser Ser 500 505 510 515 ctc agt gcc ttg caa gcc atc agt gaa ggcgtg ggg acc tct ctt tta 1702 Leu Ser Ala Leu Gln Ala Ile Ser Glu Gly ValGly Thr Ser Leu Leu 520 525 530 tct act ctg tcc tca cca ggc ccc aaa ctggat aat tct ccc aat atg 1750 Ser Thr Leu Ser Ser Pro Gly Pro Lys Leu AspAsn Ser Pro Asn Met 535 540 545 aat ata agc cag cca agt aaa gtg agt ggtcag gac tct aag agc ccc 1798 Asn Ile Ser Gln Pro Ser Lys Val Ser Gly GlnAsp Ser Lys Ser Pro 550 555 560 cta ggc tta tac tgt gaa cag aat cca gtggag agt tca gtg tgt cag 1846 Leu Gly Leu Tyr Cys Glu Gln Asn Pro Val GluSer Ser Val Cys Gln 565 570 575 tca aac agc aga gat ccc caa gtg aaa aaagaa agc aag gag agc agt 1894 Ser Asn Ser Arg Asp Pro Gln Val Lys Lys GluSer Lys Glu Ser Ser 580 585 590 595 ggg gag gtg tca gag acg ccc agg ggacct ctg gaa agc aaa ggc cac 1942 Gly Glu Val Ser Glu Thr Pro Arg Gly ProLeu Glu Ser Lys Gly His 600 605 610 aag aaa ctg ctg cag tta ctc acg tgctcc tcc gac gac cga ggc cat 1990 Lys Lys Leu Leu Gln Leu Leu Thr Cys SerSer Asp Asp Arg Gly His 615 620 625 tcc tcc ttg acc aac tct ccc ctg gatcca aac tgc aaa gac tct tcc 2038 Ser Ser Leu Thr Asn Ser Pro Leu Asp ProAsn Cys Lys Asp Ser Ser 630 635 640 gtt agt gtc acc agc ccc tct gga gtgtcc tcc tca aca tca ggg aca 2086 Val Ser Val Thr Ser Pro Ser Gly Val SerSer Ser Thr Ser Gly Thr 645 650 655 gtg tct tcc acc tcc aat gtg cat gggtct ctg ttg caa gag aaa cac 2134 Val Ser Ser Thr Ser Asn Val His Gly SerLeu Leu Gln Glu Lys His 660 665 670 675 cgg att ttg cac aag ttg ctg cagaat ggc aac tcc cca gcg gag gtc 2182 Arg Ile Leu His Lys Leu Leu Gln AsnGly Asn Ser Pro Ala Glu Val 680 685 690 gcc aag atc act gca gag gcc actggg aag gac acg agc agc act gct 2230 Ala Lys Ile Thr Ala Glu Ala Thr GlyLys Asp Thr Ser Ser Thr Ala 695 700 705 tcc tgt gga gag ggg aca acc aggcag gag cag ctg agt cct aag aag 2278 Ser Cys Gly Glu Gly Thr Thr Arg GlnGlu Gln Leu Ser Pro Lys Lys 710 715 720 aag gag aat aat gct ctg ctt agatac ctg ctg gac agg gat gac ccc 2326 Lys Glu Asn Asn Ala Leu Leu Arg TyrLeu Leu Asp Arg Asp Asp Pro 725 730 735 agt gat gtg ctt gcc aaa gag ctgcag ccc cag gcc gac agt ggg gac 2374 Ser Asp Val Leu Ala Lys Glu Leu GlnPro Gln Ala Asp Ser Gly Asp 740 745 750 755 agt aaa ctg agt cag tgc agctgc tcc acc aat ccc agc tct ggc caa 2422 Ser Lys Leu Ser Gln Cys Ser CysSer Thr Asn Pro Ser Ser Gly Gln 760 765 770 gag aaa gac ccc aaa att aagacc gag acg aac gac gag gta tcg gga 2470 Glu Lys Asp Pro Lys Ile Lys ThrGlu Thr Asn Asp Glu Val Ser Gly 775 780 785 gac ctg gat aat cta gat gccatt ctt gga gat ttg acc agt tct gac 2518 Asp Leu Asp Asn Leu Asp Ala IleLeu Gly Asp Leu Thr Ser Ser Asp 790 795 800 ttc tac aac aat cct aca aatggc ggt cac cca ggg gcc aaa cag cag 2566 Phe Tyr Asn Asn Pro Thr Asn GlyGly His Pro Gly Ala Lys Gln Gln 805 810 815 atg ttt gca gga ccg agt tctctg ggt ttg cga agt cca cag cct gtg 2614 Met Phe Ala Gly Pro Ser Ser LeuGly Leu Arg Ser Pro Gln Pro Val 820 825 830 835 cag tct gtt cgt cct ccatat aac cga gcg gtg tct ctg gat agc cct 2662 Gln Ser Val Arg Pro Pro TyrAsn Arg Ala Val Ser Leu Asp Ser Pro 840 845 850 gtg tct gtt ggc tca ggtccg cca gtg aag aat gtc agt gct ttc cct 2710 Val Ser Val Gly Ser Gly ProPro Val Lys Asn Val Ser Ala Phe Pro 855 860 865 ggg tta cca aaa cag cccata ctg gct ggg aat cca aga atg atg gat 2758 Gly Leu Pro Lys Gln Pro IleLeu Ala Gly Asn Pro Arg Met Met Asp 870 875 880 agt cag gag aat tac ggtgcc aac atg ggc cca aac aga aat gtt cct 2806 Ser Gln Glu Asn Tyr Gly AlaAsn Met Gly Pro Asn Arg Asn Val Pro 885 890 895 gtg aat ccg act tcc tccccc gga gac tgg ggc tta gct aac tca agg 2854 Val Asn Pro Thr Ser Ser ProGly Asp Trp Gly Leu Ala Asn Ser Arg 900 905 910 915 gcc agc aga atg gagcct ctg gca tca agt ccc ctg gga aga act gga 2902 Ala Ser Arg Met Glu ProLeu Ala Ser Ser Pro Leu Gly Arg Thr Gly 920 925 930 gcc gat tac agt gccact tta ccc aga cct gcc atg ggg ggc tct gtg 2950 Ala Asp Tyr Ser Ala ThrLeu Pro Arg Pro Ala Met Gly Gly Ser Val 935 940 945 cct acc ttg cca cttcgt tct aat cga ctg cca ggt gca aga cca tcg 2998 Pro Thr Leu Pro Leu ArgSer Asn Arg Leu Pro Gly Ala Arg Pro Ser 950 955 960 ttg cag caa cag cagcag caa cag cag caa cag caa caa caa cag cag 3046 Leu Gln Gln Gln Gln GlnGln Gln Gln Gln Gln Gln Gln Gln Gln Gln 965 970 975 caa cag cag cag caacag cag cag atg ctt caa atg aga act ggt gag 3094 Gln Gln Gln Gln Gln GlnGln Gln Met Leu Gln Met Arg Thr Gly Glu 980 985 990 995 att ccc atg ggaatg gga gtc aat ccn tat agc cca gca gtg cag 3139 Ile Pro Met Gly Met GlyVal Asn Pro Tyr Ser Pro Ala Val Gln 1000 1005 1010 tct aac caa cca ggttcc tgg cca gag ggc atg ctc tct atg gaa 3184 Ser Asn Gln Pro Gly Ser TrpPro Glu Gly Met Leu Ser Met Glu 1015 1020 1025 caa ggt cct cac ggg tctcaa aat agg cct ctt ctt aga aac tct 3229 Gln Gly Pro His Gly Ser Gln AsnArg Pro Leu Leu Arg Asn Ser 1030 1035 1040 ctg gat gat ctg ctt ggg ccacct tct aac gca gag ggc cag agt 3274 Leu Asp Asp Leu Leu Gly Pro Pro SerAsn Ala Glu Gly Gln Ser 1045 1050 1055 gac gag aga gct ctg ctg gac cagctg cac aca ttc ctg agc aac 3319 Asp Glu Arg Ala Leu Leu Asp Gln Leu HisThr Phe Leu Ser Asn 1060 1065 1070 aca gat gcc aca ggt ctg gag gag atcgac agg gcc ttg gga att 3364 Thr Asp Ala Thr Gly Leu Glu Glu Ile Asp ArgAla Leu Gly Ile 1075 1080 1085 cct gag ctc gtg aat cag gga caa gct ttggag tcc aaa cag gat 3409 Pro Glu Leu Val Asn Gln Gly Gln Ala Leu Glu SerLys Gln Asp 1090 1095 1100 gtt ttc caa ggc caa gaa gca gca gta atg atggat cag aag gct 3454 Val Phe Gln Gly Gln Glu Ala Ala Val Met Met Asp GlnLys Ala 1105 1110 1115 gca cta tat gga cag aca tac cca gct cag ggt cctccc ctt caa 3499 Ala Leu Tyr Gly Gln Thr Tyr Pro Ala Gln Gly Pro Pro LeuGln 1120 1125 1130 gga ggc ttt aac ctt cag gga cag tca cca tcg ttt aactct atg 3544 Gly Gly Phe Asn Leu Gln Gly Gln Ser Pro Ser Phe Asn Ser Met1135 1140 1145 atg ggt cag att agc cag caa ggc agc ttt cct ctg caa ggcatg 3589 Met Gly Gln Ile Ser Gln Gln Gly Ser Phe Pro Leu Gln Gly Met1150 1155 1160 cat cct aga gcc ggc ctc gtg aga cca agg acc aac acc ccgaag 3634 His Pro Arg Ala Gly Leu Val Arg Pro Arg Thr Asn Thr Pro Lys1165 1170 1175 cag ctg aga atg cag ctt cag cag agg cta cag ggc cag cagttt 3679 Gln Leu Arg Met Gln Leu Gln Gln Arg Leu Gln Gly Gln Gln Phe1180 1185 1190 tta aat cag agc cgg cag gca ctt gaa atg aaa atg gag aaccct 3724 Leu Asn Gln Ser Arg Gln Ala Leu Glu Met Lys Met Glu Asn Pro1195 1200 1205 gct ggc act gct gtg atg agg ccc atg atg ccc cag gct ttcttt 3769 Ala Gly Thr Ala Val Met Arg Pro Met Met Pro Gln Ala Phe Phe1210 1215 1220 aat gcc caa atg gct gcc cag cag aaa cga gag ctg atg agccat 3814 Asn Ala Gln Met Ala Ala Gln Gln Lys Arg Glu Leu Met Ser His1225 1230 1235 cac ctg cag cag cag agg atg gcg atg atg atg tca caa ccacag 3859 His Leu Gln Gln Gln Arg Met Ala Met Met Met Ser Gln Pro Gln1240 1245 1250 cct cag gcc ttc agc cca cct ccc aac gtc acc gcc tcc cccagc 3904 Pro Gln Ala Phe Ser Pro Pro Pro Asn Val Thr Ala Ser Pro Ser1255 1260 1265 atg gac ggg gtt ttg gca ggt tca gca atg ccg caa gcc cctcca 3949 Met Asp Gly Val Leu Ala Gly Ser Ala Met Pro Gln Ala Pro Pro1270 1275 1280 caa cag ttt cca tat cca gca aat tac gga acg gga caa ccacca 3994 Gln Gln Phe Pro Tyr Pro Ala Asn Tyr Gly Thr Gly Gln Pro Pro1285 1290 1295 gta gcc agc ctt tgg tcg agg ctc gag tcc tcc cag tgc aatgat 4039 Val Ala Ser Leu Trp Ser Arg Leu Glu Ser Ser Gln Cys Asn Asp1300 1305 1310 gtc atc aag aat ggg gcc ttc cca gaa tgc cat ggt gca gcatcc 4084 Val Ile Lys Asn Gly Ala Phe Pro Glu Cys His Gly Ala Ala Ser1315 1320 1325 tca gcc cac acc cat gta tca gcc ttc aga tat gaa ggg gtggcc 4129 Ser Ala His Thr His Val Ser Ala Phe Arg Tyr Glu Gly Val Ala1330 1335 1340 gtc agg gaa cct ggc cag gaa tgg ctc ctt ccc cca gca gcagtt 4174 Val Arg Glu Pro Gly Gln Glu Trp Leu Leu Pro Pro Ala Ala Val1345 1350 1355 tgc tcc cca ggg gaa ccc tgc agc cta caa cat ggt gca tatgaa 4219 Cys Ser Pro Gly Glu Pro Cys Ser Leu Gln His Gly Ala Tyr Glu1360 1365 1370 cag cag cgg tgg gca ctt ggg aca gat ggc cat gac ccc catgcc 4264 Gln Gln Arg Trp Ala Leu Gly Thr Asp Gly His Asp Pro His Ala1375 1380 1385 cat gtc tgg cat gcc cat ggg ccc cga tca gaa ata ctg ctgaca 4309 His Val Trp His Ala His Gly Pro Arg Ser Glu Ile Leu Leu Thr1390 1395 1400 tct ccc tag tgggactgac tgtacagatg acactgcaca ggatcatcag4358 Ser Pro gacgtggcgg cgagtcattg tctaagcatc cagcttggaa gcaaggccagcgtgaccagc 4418 agcggggtct gtgctgtcat ttgagcagag ctgggtctcg ctgaagcgcactgtctacct 4478 gatgccctgc ctctgtgtgg caaggtgttc tgcctcatga ggatgtgattctggagatgg 4538 ggtgttcgta agcaccgctc tcttacgtca ctcccttctg cctcgccagccaaagtcttc 4598 acgtagatct agatggctag ggtttctgtc ttgcagcact ggacgagggggcacactctg 4658 ccttctcgcg tgtcgtcagc aagttagttc gtgtcgctct cctgtccagtgcaatcagtg 4718 tttctgcgct cttgtccttt acaggtgtaa tccccaagtc tgtcgtcctagtctctcctg 4778 gtgaagtccc cgtacctgta atctcaacaa ttctcattga agtttaaatggcttttgaaa 4838 aaagggaaaa atgaaaatgg ca 4860 2 1402 PRT Mus musculusmisc_feature (3121)..(3121) “n” is any nucleotide 2 Met Ser Gly Leu GlyGlu Ser Ser Leu Asp Pro Leu Ala Ala Glu Ser 1 5 10 15 Arg Lys Arg LysLeu Pro Cys Asp Ala Pro Gly Gln Gly Leu Val Tyr 20 25 30 Ser Gly Glu LysTrp Arg Arg Glu Gln Glu Ser Lys Tyr Ile Glu Glu 35 40 45 Leu Ala Glu LeuIle Ser Ala Asn Leu Ser Asp Ile Asp Asn Phe Asn 50 55 60 Val Lys Pro AspLys Cys Ala Ile Leu Lys Glu Thr Val Arg Gln Ile 65 70 75 80 Arg Gln IleLys Glu Gln Gly Lys Thr Ile Ser Ser Asp Asp Asp Val 85 90 95 Gln Lys AlaAsp Val Ser Ser Thr Gly Gln Gly Val Ile Asp Lys Asp 100 105 110 Ser LeuGly Pro Leu Leu Leu Gln Ala Leu Asp Gly Phe Leu Phe Val 115 120 125 ValAsn Arg Asp Gly Asn Ile Val Phe Val Ser Glu Asn Val Thr Gln 130 135 140Tyr Leu Gln Tyr Lys Gln Glu Asp Leu Val Asn Thr Ser Val Tyr Ser 145 150155 160 Ile Leu His Glu Pro Arg Arg Lys Asp Phe Leu Asn Thr Tyr Gln Asn165 170 175 Pro Gln Leu Met Glu Phe Leu Gly Leu Met Arg Thr Arg Asp LysLys 180 185 190 Ala Pro Tyr Ile Leu Ile Val Arg Met Leu Met Lys Thr HisAsp Ile 195 200 205 Leu Glu Asp Val Asn Ala Ser Pro Glu Thr Arg Gln ArgTyr Glu Thr 210 215 220 Met Gln Cys Phe Ala Leu Ser Gln Pro Arg Ala MetLeu Glu Glu Gly 225 230 235 240 Glu Asp Leu Gln Cys Cys Met Ile Cys ValAla Arg Arg Val Thr Ala 245 250 255 Pro Phe Pro Ser Ser Pro Glu Ser PheIle Thr Arg His Asp Leu Ser 260 265 270 Gly Lys Val Val Asn Ile Asp ThrAsn Ser Leu Arg Ser Ser Met Arg 275 280 285 Pro Gly Phe Glu Asp Ile IleArg Arg Cys Ile Gln Arg Phe Phe Ser 290 295 300 Leu Asn Asp Gly Gln SerTrp Ser Gln Lys Arg His Tyr Gln Glu Ala 305 310 315 320 Tyr Val His GlyHis Ala Glu Thr Pro Val Tyr Arg Phe Ser Leu Ala 325 330 335 Asp Gly ThrIle Val Ser Ala Gln Thr Lys Ser Lys Leu Phe Arg Asn 340 345 350 Pro ValThr Asn Asp Arg His Gly Phe Ile Ser Thr His Phe Leu Gln 355 360 365 ArgGlu Gln Asn Gly Tyr Arg Pro Asn Pro Ile Pro Gln Asp Lys Gly 370 375 380Ile Arg Pro Pro Ala Ala Gly Cys Gly Val Ser Met Ser Pro Asn Gln 385 390395 400 Asn Val Gln Met Met Gly Ser Arg Thr Tyr Gly Val Pro Asp Pro Ser405 410 415 Asn Thr Gly Gln Met Gly Gly Ala Arg Tyr Gly Ala Ser Ser SerVal 420 425 430 Ala Ser Leu Thr Pro Gly Gln Ser Leu Gln Ser Pro Ser SerTyr Gln 435 440 445 Asn Ser Ser Tyr Gly Leu Ser Met Ser Ser Pro Pro HisGly Ser Pro 450 455 460 Gly Leu Gly Pro Asn Gln Gln Asn Ile Met Ile SerPro Arg Asn Arg 465 470 475 480 Gly Ser Pro Lys Met Ala Ser His Gln PheSer Pro Ala Ala Gly Ala 485 490 495 His Ser Pro Met Gly Pro Ser Gly AsnThr Gly Ser His Ser Phe Ser 500 505 510 Ser Ser Ser Leu Ser Ala Leu GlnAla Ile Ser Glu Gly Val Gly Thr 515 520 525 Ser Leu Leu Ser Thr Leu SerSer Pro Gly Pro Lys Leu Asp Asn Ser 530 535 540 Pro Asn Met Asn Ile SerGln Pro Ser Lys Val Ser Gly Gln Asp Ser 545 550 555 560 Lys Ser Pro LeuGly Leu Tyr Cys Glu Gln Asn Pro Val Glu Ser Ser 565 570 575 Val Cys GlnSer Asn Ser Arg Asp Pro Gln Val Lys Lys Glu Ser Lys 580 585 590 Glu SerSer Gly Glu Val Ser Glu Thr Pro Arg Gly Pro Leu Glu Ser 595 600 605 LysGly His Lys Lys Leu Leu Gln Leu Leu Thr Cys Ser Ser Asp Asp 610 615 620Arg Gly His Ser Ser Leu Thr Asn Ser Pro Leu Asp Pro Asn Cys Lys 625 630635 640 Asp Ser Ser Val Ser Val Thr Ser Pro Ser Gly Val Ser Ser Ser Thr645 650 655 Ser Gly Thr Val Ser Ser Thr Ser Asn Val His Gly Ser Leu LeuGln 660 665 670 Glu Lys His Arg Ile Leu His Lys Leu Leu Gln Asn Gly AsnSer Pro 675 680 685 Ala Glu Val Ala Lys Ile Thr Ala Glu Ala Thr Gly LysAsp Thr Ser 690 695 700 Ser Thr Ala Ser Cys Gly Glu Gly Thr Thr Arg GlnGlu Gln Leu Ser 705 710 715 720 Pro Lys Lys Lys Glu Asn Asn Ala Leu LeuArg Tyr Leu Leu Asp Arg 725 730 735 Asp Asp Pro Ser Asp Val Leu Ala LysGlu Leu Gln Pro Gln Ala Asp 740 745 750 Ser Gly Asp Ser Lys Leu Ser GlnCys Ser Cys Ser Thr Asn Pro Ser 755 760 765 Ser Gly Gln Glu Lys Asp ProLys Ile Lys Thr Glu Thr Asn Asp Glu 770 775 780 Val Ser Gly Asp Leu AspAsn Leu Asp Ala Ile Leu Gly Asp Leu Thr 785 790 795 800 Ser Ser Asp PheTyr Asn Asn Pro Thr Asn Gly Gly His Pro Gly Ala 805 810 815 Lys Gln GlnMet Phe Ala Gly Pro Ser Ser Leu Gly Leu Arg Ser Pro 820 825 830 Gln ProVal Gln Ser Val Arg Pro Pro Tyr Asn Arg Ala Val Ser Leu 835 840 845 AspSer Pro Val Ser Val Gly Ser Gly Pro Pro Val Lys Asn Val Ser 850 855 860Ala Phe Pro Gly Leu Pro Lys Gln Pro Ile Leu Ala Gly Asn Pro Arg 865 870875 880 Met Met Asp Ser Gln Glu Asn Tyr Gly Ala Asn Met Gly Pro Asn Arg885 890 895 Asn Val Pro Val Asn Pro Thr Ser Ser Pro Gly Asp Trp Gly LeuAla 900 905 910 Asn Ser Arg Ala Ser Arg Met Glu Pro Leu Ala Ser Ser ProLeu Gly 915 920 925 Arg Thr Gly Ala Asp Tyr Ser Ala Thr Leu Pro Arg ProAla Met Gly 930 935 940 Gly Ser Val Pro Thr Leu Pro Leu Arg Ser Asn ArgLeu Pro Gly Ala 945 950 955 960 Arg Pro Ser Leu Gln Gln Gln Gln Gln GlnGln Gln Gln Gln Gln Gln 965 970 975 Gln Gln Gln Gln Gln Gln Gln Gln GlnGln Gln Met Leu Gln Met Arg 980 985 990 Thr Gly Glu Ile Pro Met Gly MetGly Val Asn Pro Tyr Ser Pro Ala 995 1000 1005 Val Gln Ser Asn Gln ProGly Ser Trp Pro Glu Gly Met Leu Ser 1010 1015 1020 Met Glu Gln Gly ProHis Gly Ser Gln Asn Arg Pro Leu Leu Arg 1025 1030 1035 Asn Ser Leu AspAsp Leu Leu Gly Pro Pro Ser Asn Ala Glu Gly 1040 1045 1050 Gln Ser AspGlu Arg Ala Leu Leu Asp Gln Leu His Thr Phe Leu 1055 1060 1065 Ser AsnThr Asp Ala Thr Gly Leu Glu Glu Ile Asp Arg Ala Leu 1070 1075 1080 GlyIle Pro Glu Leu Val Asn Gln Gly Gln Ala Leu Glu Ser Lys 1085 1090 1095Gln Asp Val Phe Gln Gly Gln Glu Ala Ala Val Met Met Asp Gln 1100 11051110 Lys Ala Ala Leu Tyr Gly Gln Thr Tyr Pro Ala Gln Gly Pro Pro 11151120 1125 Leu Gln Gly Gly Phe Asn Leu Gln Gly Gln Ser Pro Ser Phe Asn1130 1135 1140 Ser Met Met Gly Gln Ile Ser Gln Gln Gly Ser Phe Pro LeuGln 1145 1150 1155 Gly Met His Pro Arg Ala Gly Leu Val Arg Pro Arg ThrAsn Thr 1160 1165 1170 Pro Lys Gln Leu Arg Met Gln Leu Gln Gln Arg LeuGln Gly Gln 1175 1180 1185 Gln Phe Leu Asn Gln Ser Arg Gln Ala Leu GluMet Lys Met Glu 1190 1195 1200 Asn Pro Ala Gly Thr Ala Val Met Arg ProMet Met Pro Gln Ala 1205 1210 1215 Phe Phe Asn Ala Gln Met Ala Ala GlnGln Lys Arg Glu Leu Met 1220 1225 1230 Ser His His Leu Gln Gln Gln ArgMet Ala Met Met Met Ser Gln 1235 1240 1245 Pro Gln Pro Gln Ala Phe SerPro Pro Pro Asn Val Thr Ala Ser 1250 1255 1260 Pro Ser Met Asp Gly ValLeu Ala Gly Ser Ala Met Pro Gln Ala 1265 1270 1275 Pro Pro Gln Gln PhePro Tyr Pro Ala Asn Tyr Gly Thr Gly Gln 1280 1285 1290 Pro Pro Val AlaSer Leu Trp Ser Arg Leu Glu Ser Ser Gln Cys 1295 1300 1305 Asn Asp ValIle Lys Asn Gly Ala Phe Pro Glu Cys His Gly Ala 1310 1315 1320 Ala SerSer Ala His Thr His Val Ser Ala Phe Arg Tyr Glu Gly 1325 1330 1335 ValAla Val Arg Glu Pro Gly Gln Glu Trp Leu Leu Pro Pro Ala 1340 1345 1350Ala Val Cys Ser Pro Gly Glu Pro Cys Ser Leu Gln His Gly Ala 1355 13601365 Tyr Glu Gln Gln Arg Trp Ala Leu Gly Thr Asp Gly His Asp Pro 13701375 1380 His Ala His Val Trp His Ala His Gly Pro Arg Ser Glu Ile Leu1385 1390 1395 Leu Thr Ser Pro 1400 3 1463 PRT Mus musculus 3 Met SerGly Met Gly Glu Asn Thr Ser Asp Pro Ser Arg Ala Glu Thr 1 5 10 15 ArgLys Arg Lys Glu Cys Pro Asp Gln Leu Gly Pro Ser Pro Lys Arg 20 25 30 SerThr Glu Lys Arg Asn Arg Glu Gln Glu Asn Lys Tyr Ile Glu Glu 35 40 45 LeuAla Glu Leu Ile Phe Ala Asn Phe Asn Asp Ile Asp Asn Phe Asn 50 55 60 PheLys Pro Asp Lys Cys Ala Ile Leu Lys Glu Thr Val Lys Gln Ile 65 70 75 80Arg Gln Ile Lys Glu Gln Glu Lys Ala Ala Ala Ala Asn Ile Asp Glu 85 90 95Val Gln Lys Ser Asp Val Ser Ser Thr Gly Gln Gly Val Ile Asp Lys 100 105110 Asp Ala Leu Gly Pro Met Met Leu Glu Ala Leu Asp Gly Phe Phe Phe 115120 125 Val Val Asn Leu Glu Gly Ser Val Val Phe Val Phe Arg Asn Val Thr130 135 140 Gln Tyr Leu Arg Tyr Asn Gln Glu Glu Leu Met Asn Lys Ser ValTyr 145 150 155 160 Ser Ile Leu His Val Gly Asp His Thr Glu Phe Val LysAsn Leu Leu 165 170 175 Pro Lys Ser Met Val Asn Gly Gly Ser Trp Ser GlyGlu Pro Pro Arg 180 185 190 Arg Ser Ser His Thr Phe Asn Cys Arg Met LeuVal Lys Pro Leu Pro 195 200 205 Asp Ser Glu Glu Glu Gly His Asp Ser GlnGlu Ala His Gln Lys Tyr 210 215 220 Glu Ala Met Gln Cys Phe Ala Val SerGln Pro Lys Ser Ile Lys Glu 225 230 235 240 Glu Gly Glu Asp Leu Gln SerCys Leu Ile Val Trp His Glu Asp Pro 245 250 255 His Glu Gly Lys Thr AsnSer Ser Leu Ile Arg Lys Leu Tyr His Pro 260 265 270 Pro Gly Pro Pro ArgGln Asp His Phe Thr Gly His Tyr His His Glu 275 280 285 Ser Arg His GluAla Gly Leu Gly Arg Ser Gly Lys Lys Asp Ala Phe 290 295 300 Arg Ser SerThr His Ser Met Lys Gly Ser Leu Tyr His Met Pro Arg 305 310 315 320 ArgHis His His Glu Val Leu Arg Gln Gly Leu Ala Phe Ser Gln Ile 325 330 335Tyr Arg Phe Ser Leu Ser Asp Gly Thr Leu Val Ala Ala Gln Thr Lys 340 345350 Ser Lys Leu Ile Arg Ser Gln Thr Thr Asn Glu Pro Gln Leu Val Ile 355360 365 Ser Leu His Met Leu His Arg Glu Gln Asn Val Cys Val Met Asn Pro370 375 380 Asp Leu Thr Gly Gln Ala Met Gly Lys Pro Leu Asn Pro Ile SerSer 385 390 395 400 Ser Ser Pro Ala His Gln Ala Leu Cys Ser Gly Asn ProGly Gln Asp 405 410 415 Met Thr Leu Ser Ser Asn Ile Asn Phe Pro Met AsnGly Pro Lys Glu 420 425 430 Gln Met Gly Met Pro Met Gly Arg Phe Gly GlySer Gly Gly Met Asn 435 440 445 His Val Ser Gly Met Gln Ala Thr Thr ProGln Gly Ser Asn Tyr Ala 450 455 460 Leu Lys Met Asn Ser Pro Ser Gln SerSer Pro Gly Met Asn Pro Gly 465 470 475 480 Gln Ala Ser Ser Val Leu SerPro Arg Gln Arg Met Ser Pro Gly Val 485 490 495 Ala Gly Ser Pro Arg IlePro Pro Ser Gln Phe Ser Pro Ala Gly Asn 500 505 510 Leu His Ser Pro ValGly Val Cys Ser Ser Thr Gly Asn Ser His Ser 515 520 525 Tyr Thr Asn SerSer Leu Asn Ala Leu Gln Ala Leu Ser Glu Gly His 530 535 540 Gly Val SerLeu Gly Ser Ser Leu Ala Ser Pro Asp Leu Lys Met Gly 545 550 555 560 AsnLeu Gln Asn Ser Pro Val Asn Met Asn Pro Pro Pro Leu Ser Lys 565 570 575Met Gly Ser Leu Asp Ser Lys Asp Cys Phe Gly Leu Tyr Gly Glu Pro 580 585590 Ser Lys Gly Thr Thr Gly Gln Ala Glu Ala Ser Cys His Pro Lys Lys 595600 605 Gln Lys Gly Pro Asn Asp Ser Ser Met Pro Gln Ala Ala Ser Gly Asp610 615 620 Arg Ala Glu Gly His Ser Arg Leu His Asp Ser Lys Gly Gln ThrLys 625 630 635 640 Leu Leu Gln Leu Leu Thr Thr Lys Ser Asp Gln Met GluPro Ser Pro 645 650 655 Leu Pro Ser Ser Leu Ser Asp Thr Asn Lys Asp SerThr Gly Ser Leu 660 665 670 Pro Gly Pro Gly Ser Thr His Gly Thr Ser LeuLys Glu Lys His Lys 675 680 685 Ile Leu His Arg Leu Leu Gln Asp Ser SerSer Pro Val Asp Leu Ala 690 695 700 Lys Leu Thr Ala Glu Ala Thr Gly LysGlu Leu Ser Gln Glu Ser Ser 705 710 715 720 Ser Thr Ala Pro Gly Ser GluVal Thr Val Lys Gln Glu Pro Ala Ser 725 730 735 Pro Lys Lys Lys Glu AsnAla Leu Leu Arg Tyr Leu Leu Asp Lys Asp 740 745 750 Asp Thr Lys Asp IleGly Leu Pro Glu Ile Thr Pro Lys Leu Glu Arg 755 760 765 Leu Asp Ser LysThr Asp Pro Ala Ser Asn Thr Lys Leu Ile Ala Met 770 775 780 Lys Thr ValLys Glu Glu Val Ser Phe Glu Pro Ser Asp Gln Pro Gly 785 790 795 800 SerGlu Leu Asp Asn Leu Glu Glu Ile Leu Asp Asp Leu Gln Asn Ser 805 810 815Gln Leu Pro Gln Leu Phe Pro Asp Thr Arg Pro Gly Ala Pro Thr Gly 820 825830 Ser Val Asp Lys Gln Ala Ile Ile Asn Asp Leu Met Gln Leu Thr Ala 835840 845 Asp Ser Ser Pro Val Pro Pro Ala Gly Ala Gln Lys Ala Ala Leu Cys850 855 860 Met Ser Gln Ser Ser Phe Asn Asn Pro Arg Pro Gly Gln Leu GlyArg 865 870 875 880 Leu Leu Pro Tyr Gln Asn Leu Pro Leu Asp Ile Thr LeuGln Ser Pro 885 890 895 Thr Gly Ala Gly Pro Phe Pro Pro Ile Arg Asn SerSer Pro Tyr Ser 900 905 910 Val Ile Pro Gln Pro Gly Met Met Gly Asn GlnGly Met Leu Gly Ser 915 920 925 Gln Gly Asn Leu Gly Asn Asn Ser Thr GlyMet Ile Gly Ser Ser Thr 930 935 940 Ser Arg Pro Ser Met Pro Ser Gly GluTrp Ala Pro Gln Ser Thr Ser 945 950 955 960 Cys Glu Ser Thr Leu Val LeuLeu Pro Leu Val Pro Arg Thr Asp Gln 965 970 975 Ser Lys Glu Ala Arg PheGly Asn Pro Thr Ala Ser Ile Pro Met Gly 980 985 990 Ala Asn Ser Gln LeuGly Gln Arg Gln Met Leu Gln Ser Gln Val Met 995 1000 1005 Asn Ile GlyPro Ser Glu Leu Glu Met Asn Met Gly Gly Pro Gln 1010 1015 1020 Tyr AsnGln Gln Gln Ala Pro Pro Asn Gln Thr Ala Pro Trp Pro 1025 1030 1035 GluSer Ile Leu Pro Ile Asp Gln Ala Ser Phe Ala Ser Gln Asn 1040 1045 1050Arg Gln Pro Phe Gly Ser Ser Pro Asp Asp Leu Leu Cys Pro His 1055 10601065 Pro Ala Ala Glu Ser Pro Ser Asp Glu Gly Ala Leu Leu Asp Gln 10701075 1080 Leu Tyr Leu Ala Leu Arg Asn Phe Asp Gly Leu Glu Glu Ile Asp1085 1090 1095 Arg Ala Leu Gly Ile Pro Glu Leu Val Ser Gln Ser Gln AlaVal 1100 1105 1110 Asp Ala Glu Gln Phe Ser Ser Gln Glu Ser Ser Ile MetLeu Glu 1115 1120 1125 Gln Lys Pro Pro Val Phe Pro Gln Gln Tyr Ala SerGln Ala Gln 1130 1135 1140 Met Ala Gln Gly Gly Tyr Asn Pro Met Gln AspPro Asn Phe His 1145 1150 1155 Thr Met Gly Gln Arg Pro Asn Tyr Thr ThrLeu Arg Met Gln Pro 1160 1165 1170 Arg Pro Gly Leu Arg Pro Thr Gly IleVal Gln Asn Gln Pro Asn 1175 1180 1185 Gln Leu Arg Leu Gln Leu Gln HisArg Leu Gln Ala Gln Gln Asn 1190 1195 1200 Arg Gln Pro Leu Met Asn GlnIle Ser Ser Val Ser Asn Val Asn 1205 1210 1215 Leu Thr Leu Arg Pro GlyVal Pro Thr Gln Ala Pro Ile Asn Ala 1220 1225 1230 Gln Met Leu Ala GlnArg Gln Arg Glu Ile Leu Asn Gln His Leu 1235 1240 1245 Arg Gln Arg GlnMet Gln Gln Gln Val Gln Gln Arg Thr Leu Met 1250 1255 1260 Met Arg GlyGln Gly Leu Asn Val Thr Pro Ser Met Val Ala Pro 1265 1270 1275 Ala GlyLeu Pro Ala Ala Met Ser Asn Pro Arg Ile Pro Gln Ala 1280 1285 1290 AsnAla Gln Gln Phe Pro Phe Pro Pro Asn Tyr Gly Ile Ser Gln 1295 1300 1305Gln Pro Asp Pro Gly Phe Thr Gly Ala Thr Thr Pro Gln Ser Pro 1310 13151320 Leu Met Ser Pro Arg Met Ala His Thr Gln Ser Pro Met Met Gln 13251330 1335 Gln Ser Gln Ala Asn Pro Ala Tyr Gln Pro Thr Ser Asp Met Asn1340 1345 1350 Gly Trp Ala Gln Gly Ser Met Gly Gly Asn Ser Met Phe SerGln 1355 1360 1365 Gln Ser Pro Pro His Phe Gly Gln Gln Ala Asn Thr SerMet Tyr 1370 1375 1380 Ser Asn Asn Met Asn Ile Ser Val Ser Met Ala ThrAsn Thr Gly 1385 1390 1395 Gly Leu Ser Ser Met Asn Gln Met Thr Gly GlnMet Ser Met Thr 1400 1405 1410 Ser Val Thr Ser Val Pro Thr Ser Gly LeuPro Ser Met Gly Pro 1415 1420 1425 Glu Gln Val Asn Asp Pro Ala Leu ArgGly Gly Asn Leu Phe Pro 1430 1435 1440 Asn Gln Leu Leu Gly Met Asp MetIle Lys Gln Glu Gly Asp Ala 1445 1450 1455 Ser Arg Lys Tyr Cys 1460 4 13PRT Mus musculus 4 Gln Thr Ser His Lys Leu Val Gln Leu Leu Thr Thr Ala 15 10 5 15 PRT Mus musculus 5 Ser Leu Thr Glu Arg His Lys Arg Leu His ArgLeu Leu Gln Glu 1 5 10 15 6 15 PRT Mus musculus 6 Ser Lys Asp His GlnLeu Leu Arg Tyr Leu Leu Asp Lys Asp Glu 1 5 10 15 7 13 PRT Mus musculus7 Ser Ser Ser Gln Leu Asp Glu Leu Leu Cys Pro Pro Thr 1 5 10 8 11 PRTMus musculus 8 Lys Ala Leu Leu Glu Gln Leu Val Ser Phe Leu 1 5 10 9 12PRT Mus musculus 9 Gln Gln Lys Ser Leu Leu Gln Gln Leu Leu Thr Glu 1 510 10 12 PRT Mus musculus 10 Lys His Lys Gln Leu Ser Glu Leu Leu Arg GlyGly 1 5 10 11 13 PRT Mus musculus 11 Ser Pro Ser Ala Leu Gln Asp Leu LeuArg Thr Leu Lys 1 5 10 12 6 PRT Mus musculus 12 Gln Leu Ala Ala Ala Ala1 5 13 6 PRT Mus musculus 13 Arg Leu Ala Ala Ala Ala 1 5 14 4 PRT Musmusculus 14 Ala Ala Ala Ala 1

What is claimed is:
 1. A substantially purified nucleic acid molecule,comprising a nucleotide sequence encoding a murine p/CIP polypeptide,wherein said nucleotide sequence encodes the same amino acid sequence asa p/CIP polypeptide with the amino acid sequence shown in FIG. 1 (SEQ ID2).
 2. A substantially purified nucleic acid molecule encoding afragment of a murine p/CIP polypeptide, which fragment comprises amurine p/CIP polypeptide comprised of SEQ ID2 (FIG. 1).
 3. Thesubstantially purified nucleic acid molecule of claim 2, comprising anucleotide sequence encoding the same amino acid sequence as a portionof a p/CIP polypeptide selected from the group consisting of about aminoacids 758-1115 of p/CIP (SEQ ID 2); about amino acids 947 to 1084 ofp/CIP (SEQ ID 2); and about amino acids 163 to 610 of p/CIP (SEQ ID 2)shown in FIG.
 1. 4. A substantially purified nucleic acid moleculeencoding a fragment of a murine p/CIP polypeptide, comprising a nuclearreceptor interaction domain of a murine p/CIP polypeptide of SEQ ID2(FIG. 1).
 5. The substantially purified nucleic acid molecule of claim4, comprising a nucleotide sequence encoding the same amino acidsequence as a portion of the p/CIP polypeptide selected from the groupconsisting of about amino acids 591 to 803 (SEQ ID 2) and about aminoacids 680 to 740 (SEQ ID 2) of p/CIP as shown in FIG.
 1. 6. Asubstantially purified p/CIP nucleotide sequence, comprising at least 50consecutive nucleotides of the nucleotide sequence shown in FIG. 1 (SEQID 1).
 7. A substantially purified p/CIP nucleotide sequence, comprisingat least 50 consecutive nucleotides encoding a fragment of thepolypeptide shown in FIG. 1 (SEQ ID 2).